CA1224432A - Dna sequences, recombinant dna molecules and processes for producing bovine growth hormone-like polypeptides in high yield - Google Patents
Dna sequences, recombinant dna molecules and processes for producing bovine growth hormone-like polypeptides in high yieldInfo
- Publication number
- CA1224432A CA1224432A CA000434118A CA434118A CA1224432A CA 1224432 A CA1224432 A CA 1224432A CA 000434118 A CA000434118 A CA 000434118A CA 434118 A CA434118 A CA 434118A CA 1224432 A CA1224432 A CA 1224432A
- Authority
- CA
- Canada
- Prior art keywords
- bgh
- growth hormone
- bovine growth
- delta
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormones [GH] (Somatotropin)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/168—Steroids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/12—Growth hormone, growth factor other than t-cell or b-cell growth factor, and growth hormone releasing factor; related peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/30—Signal or leader sequence
Abstract
DNA sequences, recombinant DNA molecules and processes for producing bovine growth hormone-like poly-peptides in high yield and the novel polypeptides produced thereby. These polypeptides are useful in improving the rate of growth of and meat production and quality in cattle.
Description
~ B36 DNA SEQUENCES, RECOMBIN~NT DNA MOLECULES
AND PROCESSES FOR PRODUCING BOVINE GROWTH
HORMONE-LIKE POLYPEPTIDES IN HIGH YIELD
h'CE~ICAL FIELD OF INVENTION
S This invention relates to novel DNA sequences, recombinant DNA molecules and processes for producing bovine growth hormone-like polypeptides in high yi~ld. More parti-cularly, it relates to novel DNA sequences expressed in appxopriate hosts in high yield and the novel bovine growth hormone-like polypeptides produced in those hosts. The DNA sequences and recombinant DNA molecules of this inven-tion are characterized in that they code for novel poly-peptides having the growth enhancin~ biological activity of bovine growth hoxmone. As will be appreciated from the disclosure to follow the DNA seguences, recombinant DNA molecules and processes of this invention may be used in the production of polypeptides useful as general anabolic agents in cattle, most especially to increase the rate of growth, weight gain and meat production in those animals.
BACK~ROUND ART
Bovine growth hormone ("BGH") is a polypeptide hormone synthesized in and secreted from the anterior lobe of the pituitary. BGH is believed to be synthesized as a precursor protein (bovine pre-growth hormone) and to be matured ~o bovine growth hormone during secretion and re-lease of the hormone into the blood stream. Moreover, it has been reported that protein fractions of natural bovine growth hormone include polypeptides that lack a number ~ ,.
of amino-terminal amino acids, but which are still biologically active.
A nucleotide coding sequence and an amino aci.d sequence of bovine growth hormone have been reported [W. L. Miller et al., J. Biol. Chem., 255, pp. 7521-24 (1980); L. Hunt and M. O. Dayhoff et al., in "Atlas Of Protein Sequence And Structure", Dayhoff ed., 5, Supp. 2, p. 139 tl976); M. Wallis, FERS Lett, 35, pp. 11-14 (1973)]. It is a polypeptide of 191 amino acids and appears to be synthesized initially as a bovine pre-growth hormone of 217 amino acids; the signal sequence of 26 amino acids being removed from the N-terminal position during synthesis and secretion [V. R. Lingappa et al., Proc. Natl.
Acad. Sci. USA, 74, pp. 2432-36 (1977)3.
Grow-th hormones are normally produced throughout the life cycle, although apparently in higher amounts during the pre-adult period. These hormones are known to promote skeletal growth, nitro gen retention, protein synthesis and to affect glucose and lipid metabolism. Accordingly, growth hormones are recognized as general anabolic agents.
Growth hormones are somewhat species speci-fic. Eowever, a growth hormone from one species maybe biologically active in another species lower in the evolutionary scale. Although the mechanism of growth hormone's activity is not well understood, it has been demonstrated that the administration of growth hormone markedly increases the rate of growth, weigh-t gain, and meat production in animals. For example, in one test, the average ra-te of weight gain in pigs receiving daily injections of purified swine growth hormone was 2.26 pounds per day (as compared to an average weight gain of 2.19 lbs/day in control pigs). More importantly, the treated pigs consumed significantly less feed per day -than the control pigs t7-03 lbs as compared to 8.40 lbs).
B.0891 -2a- ~ 2 ~ ~ ~ 3 ~
In addition, the treated pigs displayed a marked improvement in carcass guality -- the carcasses of the growth hormone-treated pigs averaged 30.57 inches in length and had 1.40 inches of backfat, while those of the control g.roup averaged 29.33 inches in length and had 1.77 inches of backfat. The chemical compo-sition o~ the edible meat t~
-3~ %
was also markedly improved in the growth hormone-treated animals -- 13.50% protein, 49.14% moisture and 36.76%
fat -- as compared to the control group -- 10.8%
protein, 39.43% moisture and 49.27% fa-t [E. J. Turman, "Some Effects Of Pituitary Anterior Growth Hormone On Swine", Thesis; Purdue University (April 1953)].
Unfortunately, the above-described improved growth and enhanced meat produc-tion in animals using growth hormone are not able to be widely realized in cattle because there is insufficient BG~ available.
Today, ~G~ is extracted from pituitary glands of ca-ttle or produced via recombinant DNA technology in appropriate hosts, e.g., W. L. Miller et al., Journal Biolo~ical Chem., 255, pp. 7521-24 (19803, European patent application 47,600 and United Kingdom patent application 2,073,245A. Plainly, the former source is not nearly adequate to provide the needed commercial ~uantities of BG~. The latter source is also not adequate because the expression yields of BGH in various hosts have been -too low to provide economically-useful or commercial quantities of BGH.
DISCLOSURE OF THE INVENTION
The present invention solves the problems referred to by providing DNA sequences that code for novel BGH-like polypeptides and by expressing those sequences in high yields in appropriate hosts to produce efficiently and economically large quantities of polypeptides displaying the grow-th enhancing, biological activity of BGH. Accordingly, by vir-tue of this invention, it is for the first time possible -to obtain polypeptides displaying the activity of BGH in commercial ~uantities for use in diverse appli-cations to increase the rate of growth of and meat production in cattle.
As will be appreciated from the disclosure to follow, the novel DNA se~uences and recombinant B.0891 ~ ~ ~J~ ~ 3 -3a-DNA molecules of this invention are capable of directing the production, in appropriate hosts, of large amounts of bovine growth B.0891 r -4- , ) hoxmone-like pol~peptides, i.e., novel pol~peptides dis-playing the growth enhancing, biological activity of BGH.
The novel polypeptides of this invention are useful, either as produced in the host or after further derivatization or modification, in compositions and methods for improving the growth rakes of and meat production in cattle.
It will accordin~ly be appreciated from the fore-going that a basic aspect o this invention is the prepara-tion of ~NA seguences which code for polypeptides display-ing the growth-enhancing biological activity of BGH, which are characterized by amino terminal deletions from the DNA se~uence coding fo~ mature bovine growth hormone t and which allow the production o~ such BG~-like product~ in at least about 100 times highex yield, and preferably at least about 1000 times higher yield, in appropriate hosts and expression vectors, than the DNA sequences coding for mature bovine growth hormone formerly employed fox such expression. Such DNA sequences comprise, for example, DNA inserts selected from the group consisting o the DNA
inserts of pBG~-Q4, p~GH-~9, pBGH-~4(Ser), pBGH-~9(Ser), and other DNA inserts charac~erized by an amino terminal deletion from the DNA sequence coding for mature bovine growth hormone, said inserts allowing the production of a bovine growth hormone-like polypeptide in at least 100 2s times higher yield than the DNA sequen e coding for mature bovine growth hormone.
BRIEF DESCRIP~ION OF THE DRAWINGS
E'igure 1 is a schema~ic outline of one embodiment of a method for preparing a recombinant DNA molecule char-acterized by a DNA sequence encoding B~.
Figures 2 and 3 display the nucleotide se~uenceof a DN~ inser~ encoding bovine pre-growth hormone, num-bered from the end o~ the dG tail to the begi~ning of the dC tail (Nucleotides 1-713). The sequence depicted includes 25 of the 26 amino acids of the putative bovine pre-growth hormone si~nal sequence (numbered amino acids -25 to -1) and 191 of ~he amino acids o~ mature bovine growth hormone (numbered amino acids 1~191) f' -5-~ 3~
Figure 4 displays one e~odiment o~ ~ process of preparing a DN~ sequence of this invention having a deletion in the amino terminal coding end of the bovine growth hormone coding region.
s Figure 5 displays one embodimen~ of a process of preparing expression vectors of this invention character-i~ed by ~NA sequences that permit high level expression o~ the novel bovine growth hormone-like polypeptides of this invention.
Fi~ure 6 displays another embodiment of a process of this invention for preparing expression vectors of this invention characterized by DNA sequenc ~ that permit high level expression of the novel bovine growth hormone-like polypeptides of this invention~
BEST MODE OF CARRYING OUT T~E INVENTION
In order that the invention herein described may be more fully understood, the following detailed descrip-tion is set forth.
In the description the following terms are emplayed:
Nucleotide -- A monomeric unit of DNA or ~NA
consisting o~ a suyar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moie~y via the glycosidic carbon (1' carbon of ~5 the pentose). That combination of a base and a sugar is called a nucleoside. Each nucleotide is characterized by its base. The four DNA bases are adenine ("~"~, guanine ("G"), cytosine ("C"~ and ~hymine ("T"). The four ~NA
bases are A, G, C and uracil ("U").
DNA Se~uence - A linear array of nucleotides connected one to the other by phosphodie~ter bonds ~etween the 3' and 5' carbons of adjacent pento es.
Codon -- A DNA se~uence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a trans lation s~art signal or a transla~ion termination signal.
For example, the nucleo~ide triplets TTA, TTG, CTT, CTC, CTA and CTG encode ~or ~he amino acid leucine ("Leu"), --6 ~
., ~ @~3~
TAG, TAA and TGA are translation s~op signals and ATG is a translation start signal.
Reading Frame -- The grouping of codons during translation of mRNA in-to amino acid sequences. During translation the proper reading frame must be maintained.
For example, the sequence GCTGGTTGTAAG may be translated in three reading frames or phases, each of which affords a different amino acid sequence:
GCT GGT TGT AAG -- A:La-Gly-Cys-Lys G CTG GTT GTA AG -- Leu-Val~Val GC TGG TTG TAA G -- Trp-Leu-(STOP) Po~ypeptide -- A linear array of amino acids connec-ted one to the other by peptide bonds between the ~--amino and carboxy groups of adjacent amino acids.
Genome -- The entire DNA of a cell or a virus.
It includes, inter alia, the genes coding for the polvpep-tides of the organism, as well as operator, promoter and ribosome binding and interaction sequences, including sequences such as the Shine-Dalgarno seguences.
Gene -- A DNA se~uence which encodes through its template or messenger RNA ("mRNA") a sequence of amino acids charac-teristic of a specific polypeptide.
~ -- The process of producing mRNA
from a gene.
Translation -- The process of producing a poly-peptide from mRNA.
Expression -- The process undergone by a DNA
sequence or gene to produce a polypeptide. It is a combi-nation of transcription and translation.
_lasmid -- A non-ch~omosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (TetR) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a "transformant".
~ ac~erial YirU ma~y of which con ist of DNA sequ~nces encap~idated in ~ pro~ein ~nv~lope or coat ~ "cap~id" ) .
Clonin~V hi~ A plas~id, phage DNA or oth~r 5 DN~ ~e~ce which i~ able to replic:a~e i~ a hos~ c~ll, which i~ ch~ac~riz~d by on~ or a small r~umber o~ e~do-nucl~a~ r~60~itiO~1 ~it~ at which 3UGh DN~ nc~Q
may be~ cu~ 1~ a de~rm~nabl~ ashio~ ~i'chou~ at~e~da~
lo~ oX a~ ~ se~tial biologic:al ~unc~:io~ of ~h~ DNP~, ~.g., 10 replicat:ion, pro~llctiola oX coat prot~ln~ 02~ lo~ o~ pxomoter or bix~di~g sit~, ~d w~ch eo~tain~ a ma~ker ~lait~le oE u~ i~ the ide~i:ific~l:ion o~ tra~formed eell~ ~ ~ . gO, toltracyclin~ re~i~ta~c~ o~ alQpi~ r~ 1ca~Ge~ A clo~i~g vehicle i~ of1:~ called ~ tor~
~ Th~ proc:e~ o obtainin~ a popula~o~
o~ org~ or DN~ s~que~c:e~ d~:ived fro~ o~ ~uch organis~
or ~e2que~ by a~ual repro~us::~lo~.
~ c~b ina~t D~ ~1 ec~-- Q r ~ m~ A. mole cule co~sisti~g o~E s~ of l~N~ ~om d~ffer~rlt g~ome~
20 which ha~e! b~e~ join~d e~d-to-end outslde of li~ing cells and hav~ ~he capacit~ ~o infec~ 30~e hos~ cQll a~d b~ main-tai~d therel~0 ~ A ~ of nus::leotide~ ~at 60~1~015 asl3l regula~ e~cpr~io~ of DN~
25 seque~ces ær ~ wlao~ op~::atavely li~d to tho~~que~ Q~. Th~y i~clude ~h~ lac ~y~t~, ~ y5t~, major o~ ator a~LdL lproms~r r~gioD~ of phag~ co~rol regio~ of fd coa~ pro~ d oth~r ~gue~r~ce~ know~ to control l:he2 ~xpre~lo~L of ~eales of proka~yo~lc: or eukaryotic ~0 c:ell ar~d their ~ e~ or c:ombinat:io~ th~r~nsfn B~I -- :Bovi~a~ growth h~ o~.
~ polyp~l?tad~ playing the growth~ ing biolo~ic21 a. tivil:y of B~3.
-a~
PR~ PARATION 0~ A ~k:COMESI~ANT DM~ MU~CULE ~AYING
R~f~rrirlg now to Figur~ 1, w¢ hav~ 3hown therein a sehematic outlin~ o~ one erabodime~t of a proces~ for 5 preparing a recombi~ DNA mol~cul~ charac~e2i2ed iZl 'chat it ha3 a DNA 5~qu~ce! coding or bs~vin~ ~rowth hormon~.
W~ ola~d th~ nc:Qdins~ bovi~ growth hor mon~ fros~ bovin~ pituitary s~ d~ by ~x~ac~i~g ~ su~
wi~ch guan dium ehlorid~ and chrom tographia~ ~ ract 10 on ~ oliso dT~ olum~ ~E3. ~dman a~d R. ~a.c33Orlald, i~
~ __ ~ ~, R. w~, ed,, 6Q, pp ~ 75 89 ( 1979 ); El. Avi~ aRd P. ~d~r, P-- c. Il tl Ac~d Sci Us~, 69, p~ O 14a8 12 ( 1972 ~ ~ ~ w~ iz~ ~ra6:~0~-at~d th~ r~ulti~g poly ~ ~NA o~ i~okin~ ucro~o 1~ ~adi~n~ ( 10-20%~ ~Bu~ll et al ., J. S;Lol . CheD~. 253, pp 0 2471~82 ~1978 ~; Wicken~æ et al ., ~ . a~O~ , 253, pp . 2~3 ~95 ( 1978 ) 3 .
we employed the~ ~ize ~ractiona~e~ poly .~ RNA containing messenger RNA ~nc:odi~g bovin~ growth ho~on~ [det~ai~ed by rabbi~ reticul oqrt~ c~ll fre~ tr~latio~ y~te~ as~ay ~El. Pelha~a and R. Jack~ora, ~r--e-., 67, ppO 247 ( 1976 ) ] ] a~ a te~plate for rever~e tran~cEiption to prepar~
~ingl~ d~d cDN~ and ~o dou~le ~ cD~ ubstan-tialily a~ d~6rlbe~dl i~ ~u~ll, ~, a~d Wick~, ~.
After l:r~a:m@~t of the douibl~-~t~a~ded cDN~ with Sl r~uclea~o to op~ haixpi~ ~cructure~, w~ add~d dC tails ~o ~ wi1~ enni~al ~a~era ~ [R. Roychoudha~ et al., ~-~, 3, pp . 101-10 ( 1976 ~ ~ . we the~
i~sert~d th~ dC-tailed eDNP. i~to a P~tI liT~a~ized, dG tailed PB~322 a~d tran~formed E_ K12 E~31ûl with ~ recircu l~iz~d ~econ~i~ant D~ ~ol~cul~ llpper ~ a~ a~ur~, 281, pp . 5S5 5g ~1981 ); Bus~ll e~ al . " ~. ~io~ 254, pp, 9~7-~3 ~ 1979 ) ], To s~lect ~ho3a clon~ having a BS:~-related DNA
in~t, we~ screened our li~rary o:E clono tgrowa on m~dia contai~n~ 15 ,ug/ml ~e~racyc:line ] wi~ r~iction e~donucleas B~1:Nl, using s~a~dard re~trictiorl c:o~ditior ,i?~. ,,_, ~ _g_ , and buffers. Since the DNA seguence reported for BGH con-tains a uni~ue BstNl site, this screening method enabled us to select two clones containing long BGH-r~lated sequences from the many other clones in our library that did not contain such seq~ences. We designated the recombi-nant DNA molecules of the two clones that we selected pBGH 108 and pB&H 220.
We isolated pBGH 108 in large ~uantity [R. D.
Klein et al., Plasmid, 3, pp. ~8-91 (19803] and determined its nucleotid~ ~equence [Ma~am and Gilbert, Proc. Natl.
Acad. Sci. USA, 74, pp. 560-64 (1977)]. We have depicted -that sequence, and its corresponding amino acid sequence in Figure 2. Our amino acid se~uence agrees with that previously reported for B~H ~Dayhoff, supra; Wallis, supra]
except a~ amino acids 47 and 6~. Our nucleotide seguence agrees with tha~ previously reported for DNA encoding BGH
[Miller, supra] excep~ at positions 177, 240 and 4~4.
The differences of position 177 and 240 do not re~ult in an amino acid cha~ge, while the difference at 454 does result in an amino acid change. This latter change is consistent with the previously report~d heterogenel~y at this position ~Wallis, ~ ].
As shown in Figures 2 and 3, the DNA se~uence of the insert of pBG~ 108 encodes 25 of the ~6 amino acids of the presumed signal sequence of bovine pre-growth hor-mone and the 191 amino acids of bovine growth hormone.
The DNA seguence also contains 65 nucleotide~ from the 3' untranslated end of BG~ (Figure 3)~
EXPRESSION OF A DNA SEQUENCE
CO~ING FOR BOVINE GROWTH HORMONE
Using pBGH 108, we isolated the DNA sequenceencoding the 191 amino acids of bovine growth hormone and constructed various expression vectors having that sequence fused directl~ to an AT& txanslation start signal and under the control of various expression control sequences to assay the level of syn~hesis of f-met-BGH (the 191 amino acids of ~GH with a me~hionine at its amino terminus~ in various E. coli host strains.
The results of those constructions and syntheses are depicted below:
Yield of BGH BG~-Like Promoter Activity (mg/l) Molecules/cell 5 trp 0.001 50 PL 0.01 ~ 1000 From these yields, we concluded that constructions that merely place an ATG start codon at the beginning of the DNA sequence coding for mature bovine growth hormone do 10 not permit the synthesis of useful amounts of BGH in E.coli.
We also constructed various expres~ion vectors having 5 amino acids of bovine pre-growth hormone (amino acids -1 to -5 in Figure 2) between the ATG start codon and the GCC encoding the first amino acid (Ala~ of mature 15 bovine growth hormone. These constructions should result in the synthesis of a product haviny the 191 amino acids of BGH with 6 amino acids fused to its amino terminus ~methionine plus the 5 amino acids from the pre~sequence of BGH).
The results of tho~e cons~ructions and syntheses are depicted below:
Yield of BG~ BGH- Like Promoter Actlvity (mg/l) Molecules~cell trp 0.01 500 25 PL 0.01 1000 From these results we concluded that merely changing the distance between the expression control sequence of the particular construction and the ATG start codon does not appreciably improve the level of expression.
30 We also concluded that varying the distance between the start codon and the first amino acid of mature bovine growth hormone does not help.
We then made a number of modifications in the PL controlled constructions, described above, to shorten 35 the distance between the BG~ coding sequence and the Shine Dalgarno sequence in the expression vector, to increase the promoter efficiency by deletions in the 99 nucleo~ide fragment carrying the Shine Dalgarno seguence (mu) of the ~11--vector, to remove the untranslated 3' dC end of -the BGH coding sequences and to increase the plasmid copy number of -the -transformed hosts. ~owever, none of these changes affected significantly the level of expression of BGH in hosts transformed with those vectors. It therefore appears that the BGH coding sequence has some inherent characteristics that pre-vent its high level expression in E.coli~*
While not wishing to be hound by theory, we believe that the low levels of expression of the BGH coding sequences in these vectors are caused by the binding of the A/G-rich sequences around and including the Shine-~algarno sequences (mu or trp) of the vectors and the T/C-rich areas at the beginning of the coding sequence for mature BGH.
E.g.: mu site AACTTAGGAGGGTTTTT
trp site ACGTAAAAAGGGTATCG
BG~I ATG GCCTTCCCA GCC ATG TCC TTG TCC
Such binding may obscure the ATG start codon and also preven-t good binding of the ribosomes to the appropriate se~uences.** Accordingly, we proposed to alter those T/C-rich areas at the beginning of the BGH coding se~uence and thus to interfere with any binding to the required A/G-rich areas of the 0 Shine-Dalgarno se~uence.
one method to affect the T/C-rich areas of the BGH coding sequence would be to employ point mutations in the early codons of the BGH coding * The low levels of observed synthesis cannot be ex-plained by degradation of BGH in E.coli., because natural BGH appears to be stable in E.coll. cell extracts.
** As further support for our theory, we have observed high levels of BGH like polypeptide expression when the BGH coding sequence is fused to a DNA sequence coding for 99 amino acids from MS2 so as to be expressed as a fused protein. However, such fused protein is not preferred for cattle treatment because the ex-tra amino acids carried by it may induce an immunogenic reaction in the treated anlmal s .
~.0891 -lla~
sequence so as to modify the T/C-rich regions but to preserve the amino acids coded for by those regions.
However, a review of the early amino acids of BGH
and the degenera-te codons coding for them reveals tha-t the T/C content of -the early codons cannot B.0891 .~
~l r~ ~ ~ 4 ~3 ~
~u~at~. A~ gl~ g d~d~ d~ m~ o~
codo~ ~ thi~ T/C rich aEea o~ ~ codi~g ~eqU~16 CONST~U~ION OF ~0~ DNA 5EQ~OES
S ~LOWI~& ~ PRODIJ~ON OF
Rg~ rE~ 10~ t~ , w~ h~Y~ d~
@~ llo~ g '~ u~ o ~12 ~ i~ 10 ~ ~dI:rI bu~ a~d 100 ,uP~ NaCl ai: 37~C
0 ~. ~2acl~ by ~d~ ~T~
15 (0.25 ~ aDd 1 ~1 r~sor~u~ a~. Aft@~ h~a~g th~ tur~
at 37~t: ~r 5 ~, w~ add~d 1 ~o~ o~ ~h2~0~ d sh~ok W~ uEiied ~L~ ~ized D~ by c~o~ato~:ra~y o~ ~5~ iora~) (@lu~ion with ~0 m~ T~ {Cl (P~ t 1 1~ ~TI~ d pool~d th~ DN~co~tal~g p~a~
(400 ~1). W~ th~ add~d 100 1ll B~l 31~1~ a~d 1 u~t of Bal 31 a~d ~cuba~ 30C o~ d~
t:Lo~ zl~d I:)l!aA, s~ b~ lly a~ le!~3cxib~
by ~ a~ta~ 0~;~@~D~ o~ ~g D~ 5~ ?r~ te~ t i N~
~ 3~, 9~ 1?1?~ 567g-~38 (L981~o W~ took 1010 ~ul aligllo~3 ~xo~ ~ a~ , 20 ~ec, 30 45 ~ ~d ~0 ~ E~ a~ o~ cl~e~
o~ d~l~tio~ ro~ ~ ariz~d DN~, W~ ga~ch~d ~e~ d~ïe~:io~ r~ac~o~ ach ali$uo~
1~ ad~ p~ a~d ~ wi~ ~
ph~ol and o~c~ s~ith C105 ,ul ~ W~2 pr~dpi~t~d ~Ghe ~wi~a~ e~ it~ (8~0 8 ~ Yi~ld- ~10 ~g ~ gach aliquot.
Si~e~ ~18 ~ e~tsiC:l~iO31 5i~ eC~TC~3 i~ pBOEIo 35 iM~t~Ala) i~ locat~d ~ to the ~ t of ~a~ DN~ 3~eqll2~1Ge e~codir~g ~ t 8s~ ~ ~hat plas~d SFi~@ 4), r~ictio~
. ~i~ N~ ~a@~ o~ s@ar~ with ~al 3~
@~a~ u~ e~ e~ g ~13-BGH and therefore will produce DN~ sequences that encode B~H-like polypeptides that lack one or more amino terminal amino acids, as compared to authentic BGH tFigure 4).
In order to select those deletions ending with a T (chosen because of the convenient location of a HindIII
site outside of the BGH coding seguence as described below), we employed the me~hod described by N. Panayotatos and K. Truong, ~ . We dissolved the DNA in 34 ~1 ~2~ added 4 ~1 10 x ~indIII buffer and 20 units ~ind III and incubated the mixture at 37C for 1 h. This restriction reaction cut the non-BGH end of the linearized DN~ at the HindIII
site (Figure 4). We then filled in the HindIII site by mixing the DNA with 5 ~1 1 mM dNTP's, 1 ~1 Klenow (1 unit) and incubated the mixture at 37C for 15 min. After frac-tiona-ting the filled~in DN~ on an agarose gel, we observed two bands: a small 100 base pair band corresponding to the minor HindIII fragment of ~he DNA and a large band corresponding to the major DNA fragmen~ (shown in Figure 4).
We eluted the maior DNA fragment from the gel, precipitated it with EtOH and collected the DNA by centri-fugation ~000 rpm, 8 min). As depicted in Figure 4, this DNA fra~ment carries at one end a filled in HindIII restric-tion site (AAGCT) and at the other end one of the various deletions in the BGH coding seguence produced by the pre-viously dessribed Bal 31 dlgestion. Plainly, recirculari-zation of the DNA will regenerate a HindIII site (AAGCTT) at the point of religation only in those cases where the Bal 31 deletion in the BG~ coding sequence terminates with a T. Therefore, we religated the DNA and cleaved it again with Hin_III to select the desired T-ending deletions (Figure 4).
To recircularize the DNA, we dissolved the EtOH
precipitate from above in 33 ~1 ~2l combined ~hat solution with 4 ~1 10 x ligation salts anA 2 ~1 T4 DNA ligase and incubated the mixture at 14C for 16 h, substantially as described by [N. Panayotatos and K. Truong, supra]. We then stopped the reaction by heating the mixture a~ 70C
for 15 min and isolated the DNA as before.
` . --1 ~ ~ , To isolate the DNA fragments sarrying the desired deletions ~i.e., those terminating in a T), we restricted the recircularized DNA from a~ove with HindIII and BamHI.
As depicted in Figure 4, such restriction allows selection of a DNA fragment containing the deleted ~GH coding sequence.
Accordingly, we dissolved the DNA in 5 ~1 10 x HindIII
huffer, added 20 units H _ III and 20 units BamHI and incubated the mixture at 37~C for 60 min. Af-ter digestion we fractiona~ed ~he mixture on an acrylamide gel and eluted the ~665 base pair fragment of desired DNA from the gel, precipitated it with EtOH and collected it by centrifuga-tion as before. This fragment (in one strand) carries at one end ~he T marking the end of the particular deletion that we effected in ~he B~H coding se~uence with Bal 31 digestion and at the other end the Bam HI cleavage residue which is located beyond the COO~ terminal end of the BGH
coding se~uence. In order to determine the nucleotide sequence of the various deletions (i,e. at which T the deletion stoppPd), we inser~ed our Bam HI-Hind III frag-ment in ind III-Bam HI-linearized pB~322 by dissolving the ~NA in 10 ~1 H20, adding 0.1 ~g ind I~I-Bam HI
digested pB~322 (1 ~1), 1.2 yl 10 x ligation buf~r and O.5 ul T4 DNA ligase and incubated the mixture at 14C
for 16 h.
After stopping the ligation, we transformed 200 ~1 of competent E. coll K12 A ~a gift of Walter Fiers) in the presence of 10 ~1 0.1 ~ MgCl2 by chilling the cells in ice for 15 min, incubating them at 42C for 2 min and incubating them at room temperature for 10 min. After adding 2 ml L-Broth, we grew the cells at 37C for 1 h and plated a 200 ~1 aliquot onto petri pla~Ps, containing L~Broth supplemented with 50 ~g/ml ampicillln. We grew our colonies at 37C for 16 h and randomly selected 24 clones from those colo~ies resistant to ampicillin~
We grew up cultures of each of these clones in 5 ml of L-Broth, supplemented with 50 ~g/ml ampicillin for 12 h at 37C and purified plasmid DNA from 1 ml of cells from each culture by the mi~iprep method described 3~
B~ H~ r~ i~ th~ 24 ~la~mid Di~s by ~d IlI~Bam ~I
ad p~ a~d~ ge~ ~Ea~o~a~A. '~
k~ 3~ D~ ha ng @gu d ~m o~ ola~ R~
aRd ~ployi~y th~ nci~g m~thod d~crib~d by nd Gilb~rt, ~,~ Seq~ci~g~ r~ al~d ~a~ on~ of th~
~t~ ue~d~ 127 tT~ 2 c:ot~a1.~ E~ OE aDI~ e~s ~9 10 ~d th~t ~o~r og ~ r~$ b~aa a~ ~uel~o1~ 112 o:~ ~C~ h~d ~a~e~ d~ t~d;lo ~ S~O~ S~
A~OW~JG ~ P~ODUCT~ON OF
BG~SKEi: :PO~ ?TI~ IN ~ YI~
ow ~ 5~ w~ h~
th~r~ o~of a ~roce~ ~or eo~ rueti~
io~ ~ec:to~ Gh~a6torized ~y a D~ ~que~c~ of thi~
i~Y~ation that allows ~ ~rodu ~io~ o ~ov~ ik~
2û pol~p~d~ i~ high y~ld.
~o att~t to exp~ DN~ ~qu~ ~ tho ~w~ cla~s~ o~ ~or~ d~ d ~ L9 ~, w~
D~ ~3~ f~ la~ ~ b~ ea~
25 Elind I~ r~ Qt wi~ X1Q110W a~ b~for~ d ~h~a cl~avir~g th~ ~ ~i~ 10 ~it~ o B~ ElI und~g ~ co~di~o~
d~scE~b~d ~ ~iou~ly., W~ iod ~ d~ r~d r~lat:~d ~ ~ag~ o~ a 5% ~by w~i~h~ ~oly~e~l~d~
~ Lo Th:~ ~ag~ ~
~e~a~d ~d ~ X~
fi~1~g ~ ~d III ~ a~d th~ ~a1 T ~
~UG~ ~de~ 104 Of F19~ 2 ~9 )O~ aC1~dQ 89 Of E?~ B 2 ~h43 ~, T~ ~r~ OU~ ~S~O~ ~re~ t~ ~:@~ 3 ~3 3 5 ~ W~ a~q~d t~ a~iea1 ~ W~ ~ad p~eviously e~ploy~ to e~r~ BS~ ~equ~c~3 u~d~s th~
co~ol o~ th~ rolaoter (~.g., p~ (Me~t~DAla~) ~ith Nc~ I, fillod i:ll th~ ~a~ wi~ ~ealow a~ el~a~e~d ~ ear^
~16~
,.
i2~dl D~ with B~ El~ u~i~g ~:o~Ye~tio~al eo~ditio~ aDd buff~r$. Thi~ frag~t carrle~ i~ o~ d at o~ 2~1S3 th~ ~e~ned C~TG ~ro~ ~ filli~y i~ of the Nco I ~it~
a~d a~ th~ o~r ~d tho r~idu~ ~o~ Bam ~I clea~a~
S ~Figur~ 53. ~ ~er~ combi~ed 0.2 l~g of th~ prepared ve~tor (2 yl) a~d O.S ~g of th~ p~epæ~d D~ i~ of li~atia~
bu~ d 0.5 ~1 T~ D~ liga~ ~d iIlcubat~d ~m at 1~C
or 16 h.
ixcu~ 2a~:io~l p~:oducQI~ ~0 ~ 0~8, 1~ re~p~cti~ly. I~ C1rl@ ~e ~us:leo~d~ s~qu~c~ be~ g with th~ ATG is A~TT:: f~llow~d by ~o add 6 oi~ ~
BC~ coding ~ c~ of Fis~ 2 ( 2~ ~4 ~ s~r ~ ~ ( Figur~ S ) .
~n ~ o~er th~ ~ucl~o~d~ $e~e~c~ be~q ~i~ th~
ATG i~ GC~ ollowad by a~i~o acid 11 of th~ B~ cod~
15 i~g sQgue~c~ o Figu~ 2 (p~-~9(So~)) (F~ S~ re-fo~, i~ th~ fi~t co~t~uctio~, t~ GOdiIl~ Ee~iO~l fQ:~
the i~ir~t four a~o acid~ o Dla~ BGE~ ha~ b~ r~noYed, th~ codo~ AGC (s~r~e) ix~ort~d a~d ~ codiD~g r~io~ for t~ fifth ~o acid of ~a~ BGEI ~odified ro~ ~TG to 20 ~TG cha~gi~g i~ ~o acid fro~ m~thio~ to leuci~e.
other co~uct:io~ a pr~e~red constructio~ of e$tio~ th@ eo~i~q r~gio~ Xor th~ irst ~o a~o acids o mab~r~ BG8 ha~ be~ ~ red, 1~ eo~o~ e~e~
i~dl a~d th~ coLi~ regio~ ~ th~ te~th ~c~ a::id 25 of ~atur~ odlifi~d rom CT~ ~:o TT~, a ~go that do~3 not a~ct its am~no ~cid (leuci~ r@~l:~t ~h~ f:Lr9t constructio~ should expr~ ~ a pro~ia o ~ ollowi~g ~equ~c~ ~et-S~ r.eu~o acid~ 6-191 of ~a~r@ B~ a~d th~ o'ch~ co~uctio~ ~hould ~pr~$~ a ~otei~ of the 30 follo~ g ~gue~c~ ~e~-ser-~ao aci~10~191 of mature BG8~* Ac~ordi~ly, w~ ha~ de~ignat~d ~ ~o v~ctoæ~
pBY~-~4 ( S~r ~ 9 ( S~x ) ( Figur~ 5 ~ t re~ Cl iYe!ly .
* Of cou~e, i~ should be under~oo~ t~a~ f~t ~5 may be remov~d froDa khe~ poly~epl:ide~ eith2r d~ g eacpres-sio~ or 511b~;el~1@I3.~ her~to to produce polyp~p~ides of ~h~
formllla 5~r-L~u~P~ to A~g~ o B~l and S~r ~loto o ~GEI, re~poc:tiY~ly. rrl ~act, sub~egu~t a~o acid - ~e~u~c~ of BOEl~(S~r) rev~al~d t~a~ f~e~ wa~
. j` 4~ cle~ed ~1~o~ du~i~g o~gpre~sioIl o~ ~Lrifieatio~O
~ 2 We employed the recircularized plasmids to trans-form E. coli K12 A, as descrlbed be~ore, again selecting those colonies that were resis~ant to ampicillin~ We then grew up cultures of 12 of the ampicillin resistant clones, isolated their plasmid DNA ~y the miniprep method, described previously, and si2ed the inserts by Pst I/Eco RI restric-tion using conventional conditions. From the results o~
the sizing, we selec~ed that plasmid DNA having the appro-priate insext. We then used this DNA to transform E. coli MC 1061, substantially as described in G. Nv Buell et al., J. Biol. Chem. 254, pp. 9279-83 ~1979 ) . We also transformed the cells with pcI~57 (a gif~ of W~ Fiers).* We selected cells transformed with our BGH rPlated plasmid ~N~ and pcI~57 by growing khe colonies fox 16 h at 30C in L-Broth, supplemented with ampicillin, as before, and kanamycin ~40 ~g/ml).
We then randomly picked ampicillin and kanamycin-resis~a~t colonies and grew them overnight in 5 ml L-Broth, supplemented as before with ampicillin and kanamycin, at 30C. We diluted the cultures with 25 ml of ~-Broth at 42C and allowed the cells to grow at 42C for 2 h (O.D.=l).*~
To isolate the BGH-like polypeptides produced by the ~ransformed cells, we took 1 ml of cells and spun them down (8000 rpm, 4 min) added 50 ~1 Laemmli buffer ~1% SDS~ [Laemmli, Nature, 2~7, pp. 681 et seg. (1970)], ~oiled the mixture for 15 min, and again centrifuged the c~118 ( ~000 rpm, 4 min) to remove the cellular debris.
We assayed the supernatant by radioimmunoassay for BGH
using rabbit an~isera ~o authentic BGHo ***
* pcI857 is a small plasmid that carries a temperature-sensitive P~ repressor and the gene coding for kanam~cin resistance.
** Thes~ are the identical growth conditions employed previously for P ~controlled expression of BG~ using the DNA sequence cod~n~ for mature BG~.
*** This is -~he identical isolation method and assay pre-viously employed for P -controlled expression of BGH using the DNA se~uence codin~ for mature BGH.
~2 ~ ~ ~3 ~
We obtained the following expression levels of BGH~like polypeptides:
Yield of BGH B~H-like Deletion Activity_(mg/l) Molecules/cell ~4~Ser~ 30.0 1.5 x 106 ~9(Ser) 100 5.0 x 106 We also used the ~4(Ser) and ~9(Ser) DNA sequences in the trp-containing vector previously used to express BGH using the DNA sequence coding for mature BGH. In those constructions, we obtained the following expression levels:
Yield of BGH BGH like Deletion Actlvity (mg~ Molecules/cell ~4(~er) 0.15 8000 ~9(Ser) 0.50 50000 Finally, we manipulated our constructions to delete fxom the constructions the AGC codon (coding for serine) that had been added by the Hind III fill-in. The AGC deleted constructions pBGH-~4 and pBGH-~9 are the more preferred constructions of our invention, with pBGH-a9 ~0 being the most preferred.* To make these constructions, we modified the fill-in of the Hind III site of the BGH-related sequence, described previously, so that only dATP
was present in the mixture. This modification added only a single A during fill-in (Figure 6). We then diluted the reaction mixture 10~fold with Sl ~uf~er ~30 mM NaOAc (pH 4.5), 300 mM NaCl, 3 mM ZnCl2~, added 2 units/~g DNA
of Sl and incubated the mixture at 37C for 30 min to cleave off the unfilled DNA overhang (Fisure 6). We then stopped the reaction with phenol and proceeded as before to restrict the DNA fragmen~ with Bam HI and insert it into the desired expression vector. Using -these vectors, we observed the following expression levels:
* Again the amino terminal f-Met produced on expression of these constructions could be removed from these BGH-related pol~peptides either during expression or subsequent thereto. Because of our experience with the actual amino acid sequence of BGH-~9(Ser), we e~pect -that the f-Met is removed either during expression or purification.
Yield of BGH BGH-like Deletion Promoter Activit~ m~/l Molecules/cell ~9 PL 100 5 x 106 We have also used the ~4 and ag DNA sequences of this inven-tion, as well as a BGH-~l cons-truction prepared by -the methods of this invention, under PL
control (with a mu-derived ribosome binding site, as before) to transform various E. coli hosts in order -to compare expression levels:
Deletion Host~- 12 Yield3 ~1 MC 1061 2 x 104 HB 101 10 x 104 PR 7 30 x 104 a4 MC 1061 3.6 x 105 HB 101 6.0 x 106 PR 7 13 x 105 ~9 MC 1061 1.4 x 106 HB 101 1.1 x 106 PR 7 1.5 x 106 Met-Ala-BGH MC 1061 7.5 ~ 10 (control) PR 7 20 x 102 Therefore, DNA sequences and recombinant DNA molecules characteri~ed by them, having portions of the B~H coding sequence deleted permit the produc-tion of bovine growth hormone-like polypeptides in high yield: at least 100 times higher than DNA
sequences coding for mature bovine growth hormones in the same expression vectors and hosts, and prefer-1 All hosts carried the pcI857 repressor plasmid.
AND PROCESSES FOR PRODUCING BOVINE GROWTH
HORMONE-LIKE POLYPEPTIDES IN HIGH YIELD
h'CE~ICAL FIELD OF INVENTION
S This invention relates to novel DNA sequences, recombinant DNA molecules and processes for producing bovine growth hormone-like polypeptides in high yi~ld. More parti-cularly, it relates to novel DNA sequences expressed in appxopriate hosts in high yield and the novel bovine growth hormone-like polypeptides produced in those hosts. The DNA sequences and recombinant DNA molecules of this inven-tion are characterized in that they code for novel poly-peptides having the growth enhancin~ biological activity of bovine growth hoxmone. As will be appreciated from the disclosure to follow the DNA seguences, recombinant DNA molecules and processes of this invention may be used in the production of polypeptides useful as general anabolic agents in cattle, most especially to increase the rate of growth, weight gain and meat production in those animals.
BACK~ROUND ART
Bovine growth hormone ("BGH") is a polypeptide hormone synthesized in and secreted from the anterior lobe of the pituitary. BGH is believed to be synthesized as a precursor protein (bovine pre-growth hormone) and to be matured ~o bovine growth hormone during secretion and re-lease of the hormone into the blood stream. Moreover, it has been reported that protein fractions of natural bovine growth hormone include polypeptides that lack a number ~ ,.
of amino-terminal amino acids, but which are still biologically active.
A nucleotide coding sequence and an amino aci.d sequence of bovine growth hormone have been reported [W. L. Miller et al., J. Biol. Chem., 255, pp. 7521-24 (1980); L. Hunt and M. O. Dayhoff et al., in "Atlas Of Protein Sequence And Structure", Dayhoff ed., 5, Supp. 2, p. 139 tl976); M. Wallis, FERS Lett, 35, pp. 11-14 (1973)]. It is a polypeptide of 191 amino acids and appears to be synthesized initially as a bovine pre-growth hormone of 217 amino acids; the signal sequence of 26 amino acids being removed from the N-terminal position during synthesis and secretion [V. R. Lingappa et al., Proc. Natl.
Acad. Sci. USA, 74, pp. 2432-36 (1977)3.
Grow-th hormones are normally produced throughout the life cycle, although apparently in higher amounts during the pre-adult period. These hormones are known to promote skeletal growth, nitro gen retention, protein synthesis and to affect glucose and lipid metabolism. Accordingly, growth hormones are recognized as general anabolic agents.
Growth hormones are somewhat species speci-fic. Eowever, a growth hormone from one species maybe biologically active in another species lower in the evolutionary scale. Although the mechanism of growth hormone's activity is not well understood, it has been demonstrated that the administration of growth hormone markedly increases the rate of growth, weigh-t gain, and meat production in animals. For example, in one test, the average ra-te of weight gain in pigs receiving daily injections of purified swine growth hormone was 2.26 pounds per day (as compared to an average weight gain of 2.19 lbs/day in control pigs). More importantly, the treated pigs consumed significantly less feed per day -than the control pigs t7-03 lbs as compared to 8.40 lbs).
B.0891 -2a- ~ 2 ~ ~ ~ 3 ~
In addition, the treated pigs displayed a marked improvement in carcass guality -- the carcasses of the growth hormone-treated pigs averaged 30.57 inches in length and had 1.40 inches of backfat, while those of the control g.roup averaged 29.33 inches in length and had 1.77 inches of backfat. The chemical compo-sition o~ the edible meat t~
-3~ %
was also markedly improved in the growth hormone-treated animals -- 13.50% protein, 49.14% moisture and 36.76%
fat -- as compared to the control group -- 10.8%
protein, 39.43% moisture and 49.27% fa-t [E. J. Turman, "Some Effects Of Pituitary Anterior Growth Hormone On Swine", Thesis; Purdue University (April 1953)].
Unfortunately, the above-described improved growth and enhanced meat produc-tion in animals using growth hormone are not able to be widely realized in cattle because there is insufficient BG~ available.
Today, ~G~ is extracted from pituitary glands of ca-ttle or produced via recombinant DNA technology in appropriate hosts, e.g., W. L. Miller et al., Journal Biolo~ical Chem., 255, pp. 7521-24 (19803, European patent application 47,600 and United Kingdom patent application 2,073,245A. Plainly, the former source is not nearly adequate to provide the needed commercial ~uantities of BG~. The latter source is also not adequate because the expression yields of BGH in various hosts have been -too low to provide economically-useful or commercial quantities of BGH.
DISCLOSURE OF THE INVENTION
The present invention solves the problems referred to by providing DNA sequences that code for novel BGH-like polypeptides and by expressing those sequences in high yields in appropriate hosts to produce efficiently and economically large quantities of polypeptides displaying the grow-th enhancing, biological activity of BGH. Accordingly, by vir-tue of this invention, it is for the first time possible -to obtain polypeptides displaying the activity of BGH in commercial ~uantities for use in diverse appli-cations to increase the rate of growth of and meat production in cattle.
As will be appreciated from the disclosure to follow, the novel DNA se~uences and recombinant B.0891 ~ ~ ~J~ ~ 3 -3a-DNA molecules of this invention are capable of directing the production, in appropriate hosts, of large amounts of bovine growth B.0891 r -4- , ) hoxmone-like pol~peptides, i.e., novel pol~peptides dis-playing the growth enhancing, biological activity of BGH.
The novel polypeptides of this invention are useful, either as produced in the host or after further derivatization or modification, in compositions and methods for improving the growth rakes of and meat production in cattle.
It will accordin~ly be appreciated from the fore-going that a basic aspect o this invention is the prepara-tion of ~NA seguences which code for polypeptides display-ing the growth-enhancing biological activity of BGH, which are characterized by amino terminal deletions from the DNA se~uence coding fo~ mature bovine growth hormone t and which allow the production o~ such BG~-like product~ in at least about 100 times highex yield, and preferably at least about 1000 times higher yield, in appropriate hosts and expression vectors, than the DNA sequences coding for mature bovine growth hormone formerly employed fox such expression. Such DNA sequences comprise, for example, DNA inserts selected from the group consisting o the DNA
inserts of pBG~-Q4, p~GH-~9, pBGH-~4(Ser), pBGH-~9(Ser), and other DNA inserts charac~erized by an amino terminal deletion from the DNA sequence coding for mature bovine growth hormone, said inserts allowing the production of a bovine growth hormone-like polypeptide in at least 100 2s times higher yield than the DNA sequen e coding for mature bovine growth hormone.
BRIEF DESCRIP~ION OF THE DRAWINGS
E'igure 1 is a schema~ic outline of one embodiment of a method for preparing a recombinant DNA molecule char-acterized by a DNA sequence encoding B~.
Figures 2 and 3 display the nucleotide se~uenceof a DN~ inser~ encoding bovine pre-growth hormone, num-bered from the end o~ the dG tail to the begi~ning of the dC tail (Nucleotides 1-713). The sequence depicted includes 25 of the 26 amino acids of the putative bovine pre-growth hormone si~nal sequence (numbered amino acids -25 to -1) and 191 of ~he amino acids o~ mature bovine growth hormone (numbered amino acids 1~191) f' -5-~ 3~
Figure 4 displays one e~odiment o~ ~ process of preparing a DN~ sequence of this invention having a deletion in the amino terminal coding end of the bovine growth hormone coding region.
s Figure 5 displays one embodimen~ of a process of preparing expression vectors of this invention character-i~ed by ~NA sequences that permit high level expression o~ the novel bovine growth hormone-like polypeptides of this invention.
Fi~ure 6 displays another embodiment of a process of this invention for preparing expression vectors of this invention characterized by DNA sequenc ~ that permit high level expression of the novel bovine growth hormone-like polypeptides of this invention~
BEST MODE OF CARRYING OUT T~E INVENTION
In order that the invention herein described may be more fully understood, the following detailed descrip-tion is set forth.
In the description the following terms are emplayed:
Nucleotide -- A monomeric unit of DNA or ~NA
consisting o~ a suyar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moie~y via the glycosidic carbon (1' carbon of ~5 the pentose). That combination of a base and a sugar is called a nucleoside. Each nucleotide is characterized by its base. The four DNA bases are adenine ("~"~, guanine ("G"), cytosine ("C"~ and ~hymine ("T"). The four ~NA
bases are A, G, C and uracil ("U").
DNA Se~uence - A linear array of nucleotides connected one to the other by phosphodie~ter bonds ~etween the 3' and 5' carbons of adjacent pento es.
Codon -- A DNA se~uence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a trans lation s~art signal or a transla~ion termination signal.
For example, the nucleo~ide triplets TTA, TTG, CTT, CTC, CTA and CTG encode ~or ~he amino acid leucine ("Leu"), --6 ~
., ~ @~3~
TAG, TAA and TGA are translation s~op signals and ATG is a translation start signal.
Reading Frame -- The grouping of codons during translation of mRNA in-to amino acid sequences. During translation the proper reading frame must be maintained.
For example, the sequence GCTGGTTGTAAG may be translated in three reading frames or phases, each of which affords a different amino acid sequence:
GCT GGT TGT AAG -- A:La-Gly-Cys-Lys G CTG GTT GTA AG -- Leu-Val~Val GC TGG TTG TAA G -- Trp-Leu-(STOP) Po~ypeptide -- A linear array of amino acids connec-ted one to the other by peptide bonds between the ~--amino and carboxy groups of adjacent amino acids.
Genome -- The entire DNA of a cell or a virus.
It includes, inter alia, the genes coding for the polvpep-tides of the organism, as well as operator, promoter and ribosome binding and interaction sequences, including sequences such as the Shine-Dalgarno seguences.
Gene -- A DNA se~uence which encodes through its template or messenger RNA ("mRNA") a sequence of amino acids charac-teristic of a specific polypeptide.
~ -- The process of producing mRNA
from a gene.
Translation -- The process of producing a poly-peptide from mRNA.
Expression -- The process undergone by a DNA
sequence or gene to produce a polypeptide. It is a combi-nation of transcription and translation.
_lasmid -- A non-ch~omosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (TetR) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a "transformant".
~ ac~erial YirU ma~y of which con ist of DNA sequ~nces encap~idated in ~ pro~ein ~nv~lope or coat ~ "cap~id" ) .
Clonin~V hi~ A plas~id, phage DNA or oth~r 5 DN~ ~e~ce which i~ able to replic:a~e i~ a hos~ c~ll, which i~ ch~ac~riz~d by on~ or a small r~umber o~ e~do-nucl~a~ r~60~itiO~1 ~it~ at which 3UGh DN~ nc~Q
may be~ cu~ 1~ a de~rm~nabl~ ashio~ ~i'chou~ at~e~da~
lo~ oX a~ ~ se~tial biologic:al ~unc~:io~ of ~h~ DNP~, ~.g., 10 replicat:ion, pro~llctiola oX coat prot~ln~ 02~ lo~ o~ pxomoter or bix~di~g sit~, ~d w~ch eo~tain~ a ma~ker ~lait~le oE u~ i~ the ide~i:ific~l:ion o~ tra~formed eell~ ~ ~ . gO, toltracyclin~ re~i~ta~c~ o~ alQpi~ r~ 1ca~Ge~ A clo~i~g vehicle i~ of1:~ called ~ tor~
~ Th~ proc:e~ o obtainin~ a popula~o~
o~ org~ or DN~ s~que~c:e~ d~:ived fro~ o~ ~uch organis~
or ~e2que~ by a~ual repro~us::~lo~.
~ c~b ina~t D~ ~1 ec~-- Q r ~ m~ A. mole cule co~sisti~g o~E s~ of l~N~ ~om d~ffer~rlt g~ome~
20 which ha~e! b~e~ join~d e~d-to-end outslde of li~ing cells and hav~ ~he capacit~ ~o infec~ 30~e hos~ cQll a~d b~ main-tai~d therel~0 ~ A ~ of nus::leotide~ ~at 60~1~015 asl3l regula~ e~cpr~io~ of DN~
25 seque~ces ær ~ wlao~ op~::atavely li~d to tho~~que~ Q~. Th~y i~clude ~h~ lac ~y~t~, ~ y5t~, major o~ ator a~LdL lproms~r r~gioD~ of phag~ co~rol regio~ of fd coa~ pro~ d oth~r ~gue~r~ce~ know~ to control l:he2 ~xpre~lo~L of ~eales of proka~yo~lc: or eukaryotic ~0 c:ell ar~d their ~ e~ or c:ombinat:io~ th~r~nsfn B~I -- :Bovi~a~ growth h~ o~.
~ polyp~l?tad~ playing the growth~ ing biolo~ic21 a. tivil:y of B~3.
-a~
PR~ PARATION 0~ A ~k:COMESI~ANT DM~ MU~CULE ~AYING
R~f~rrirlg now to Figur~ 1, w¢ hav~ 3hown therein a sehematic outlin~ o~ one erabodime~t of a proces~ for 5 preparing a recombi~ DNA mol~cul~ charac~e2i2ed iZl 'chat it ha3 a DNA 5~qu~ce! coding or bs~vin~ ~rowth hormon~.
W~ ola~d th~ nc:Qdins~ bovi~ growth hor mon~ fros~ bovin~ pituitary s~ d~ by ~x~ac~i~g ~ su~
wi~ch guan dium ehlorid~ and chrom tographia~ ~ ract 10 on ~ oliso dT~ olum~ ~E3. ~dman a~d R. ~a.c33Orlald, i~
~ __ ~ ~, R. w~, ed,, 6Q, pp ~ 75 89 ( 1979 ); El. Avi~ aRd P. ~d~r, P-- c. Il tl Ac~d Sci Us~, 69, p~ O 14a8 12 ( 1972 ~ ~ ~ w~ iz~ ~ra6:~0~-at~d th~ r~ulti~g poly ~ ~NA o~ i~okin~ ucro~o 1~ ~adi~n~ ( 10-20%~ ~Bu~ll et al ., J. S;Lol . CheD~. 253, pp 0 2471~82 ~1978 ~; Wicken~æ et al ., ~ . a~O~ , 253, pp . 2~3 ~95 ( 1978 ) 3 .
we employed the~ ~ize ~ractiona~e~ poly .~ RNA containing messenger RNA ~nc:odi~g bovin~ growth ho~on~ [det~ai~ed by rabbi~ reticul oqrt~ c~ll fre~ tr~latio~ y~te~ as~ay ~El. Pelha~a and R. Jack~ora, ~r--e-., 67, ppO 247 ( 1976 ) ] ] a~ a te~plate for rever~e tran~cEiption to prepar~
~ingl~ d~d cDN~ and ~o dou~le ~ cD~ ubstan-tialily a~ d~6rlbe~dl i~ ~u~ll, ~, a~d Wick~, ~.
After l:r~a:m@~t of the douibl~-~t~a~ded cDN~ with Sl r~uclea~o to op~ haixpi~ ~cructure~, w~ add~d dC tails ~o ~ wi1~ enni~al ~a~era ~ [R. Roychoudha~ et al., ~-~, 3, pp . 101-10 ( 1976 ~ ~ . we the~
i~sert~d th~ dC-tailed eDNP. i~to a P~tI liT~a~ized, dG tailed PB~322 a~d tran~formed E_ K12 E~31ûl with ~ recircu l~iz~d ~econ~i~ant D~ ~ol~cul~ llpper ~ a~ a~ur~, 281, pp . 5S5 5g ~1981 ); Bus~ll e~ al . " ~. ~io~ 254, pp, 9~7-~3 ~ 1979 ) ], To s~lect ~ho3a clon~ having a BS:~-related DNA
in~t, we~ screened our li~rary o:E clono tgrowa on m~dia contai~n~ 15 ,ug/ml ~e~racyc:line ] wi~ r~iction e~donucleas B~1:Nl, using s~a~dard re~trictiorl c:o~ditior ,i?~. ,,_, ~ _g_ , and buffers. Since the DNA seguence reported for BGH con-tains a uni~ue BstNl site, this screening method enabled us to select two clones containing long BGH-r~lated sequences from the many other clones in our library that did not contain such seq~ences. We designated the recombi-nant DNA molecules of the two clones that we selected pBGH 108 and pB&H 220.
We isolated pBGH 108 in large ~uantity [R. D.
Klein et al., Plasmid, 3, pp. ~8-91 (19803] and determined its nucleotid~ ~equence [Ma~am and Gilbert, Proc. Natl.
Acad. Sci. USA, 74, pp. 560-64 (1977)]. We have depicted -that sequence, and its corresponding amino acid sequence in Figure 2. Our amino acid se~uence agrees with that previously reported for B~H ~Dayhoff, supra; Wallis, supra]
except a~ amino acids 47 and 6~. Our nucleotide seguence agrees with tha~ previously reported for DNA encoding BGH
[Miller, supra] excep~ at positions 177, 240 and 4~4.
The differences of position 177 and 240 do not re~ult in an amino acid cha~ge, while the difference at 454 does result in an amino acid change. This latter change is consistent with the previously report~d heterogenel~y at this position ~Wallis, ~ ].
As shown in Figures 2 and 3, the DNA se~uence of the insert of pBG~ 108 encodes 25 of the ~6 amino acids of the presumed signal sequence of bovine pre-growth hor-mone and the 191 amino acids of bovine growth hormone.
The DNA seguence also contains 65 nucleotide~ from the 3' untranslated end of BG~ (Figure 3)~
EXPRESSION OF A DNA SEQUENCE
CO~ING FOR BOVINE GROWTH HORMONE
Using pBGH 108, we isolated the DNA sequenceencoding the 191 amino acids of bovine growth hormone and constructed various expression vectors having that sequence fused directl~ to an AT& txanslation start signal and under the control of various expression control sequences to assay the level of syn~hesis of f-met-BGH (the 191 amino acids of ~GH with a me~hionine at its amino terminus~ in various E. coli host strains.
The results of those constructions and syntheses are depicted below:
Yield of BGH BG~-Like Promoter Activity (mg/l) Molecules/cell 5 trp 0.001 50 PL 0.01 ~ 1000 From these yields, we concluded that constructions that merely place an ATG start codon at the beginning of the DNA sequence coding for mature bovine growth hormone do 10 not permit the synthesis of useful amounts of BGH in E.coli.
We also constructed various expres~ion vectors having 5 amino acids of bovine pre-growth hormone (amino acids -1 to -5 in Figure 2) between the ATG start codon and the GCC encoding the first amino acid (Ala~ of mature 15 bovine growth hormone. These constructions should result in the synthesis of a product haviny the 191 amino acids of BGH with 6 amino acids fused to its amino terminus ~methionine plus the 5 amino acids from the pre~sequence of BGH).
The results of tho~e cons~ructions and syntheses are depicted below:
Yield of BG~ BGH- Like Promoter Actlvity (mg/l) Molecules~cell trp 0.01 500 25 PL 0.01 1000 From these results we concluded that merely changing the distance between the expression control sequence of the particular construction and the ATG start codon does not appreciably improve the level of expression.
30 We also concluded that varying the distance between the start codon and the first amino acid of mature bovine growth hormone does not help.
We then made a number of modifications in the PL controlled constructions, described above, to shorten 35 the distance between the BG~ coding sequence and the Shine Dalgarno sequence in the expression vector, to increase the promoter efficiency by deletions in the 99 nucleo~ide fragment carrying the Shine Dalgarno seguence (mu) of the ~11--vector, to remove the untranslated 3' dC end of -the BGH coding sequences and to increase the plasmid copy number of -the -transformed hosts. ~owever, none of these changes affected significantly the level of expression of BGH in hosts transformed with those vectors. It therefore appears that the BGH coding sequence has some inherent characteristics that pre-vent its high level expression in E.coli~*
While not wishing to be hound by theory, we believe that the low levels of expression of the BGH coding sequences in these vectors are caused by the binding of the A/G-rich sequences around and including the Shine-~algarno sequences (mu or trp) of the vectors and the T/C-rich areas at the beginning of the coding sequence for mature BGH.
E.g.: mu site AACTTAGGAGGGTTTTT
trp site ACGTAAAAAGGGTATCG
BG~I ATG GCCTTCCCA GCC ATG TCC TTG TCC
Such binding may obscure the ATG start codon and also preven-t good binding of the ribosomes to the appropriate se~uences.** Accordingly, we proposed to alter those T/C-rich areas at the beginning of the BGH coding se~uence and thus to interfere with any binding to the required A/G-rich areas of the 0 Shine-Dalgarno se~uence.
one method to affect the T/C-rich areas of the BGH coding sequence would be to employ point mutations in the early codons of the BGH coding * The low levels of observed synthesis cannot be ex-plained by degradation of BGH in E.coli., because natural BGH appears to be stable in E.coll. cell extracts.
** As further support for our theory, we have observed high levels of BGH like polypeptide expression when the BGH coding sequence is fused to a DNA sequence coding for 99 amino acids from MS2 so as to be expressed as a fused protein. However, such fused protein is not preferred for cattle treatment because the ex-tra amino acids carried by it may induce an immunogenic reaction in the treated anlmal s .
~.0891 -lla~
sequence so as to modify the T/C-rich regions but to preserve the amino acids coded for by those regions.
However, a review of the early amino acids of BGH
and the degenera-te codons coding for them reveals tha-t the T/C content of -the early codons cannot B.0891 .~
~l r~ ~ ~ 4 ~3 ~
~u~at~. A~ gl~ g d~d~ d~ m~ o~
codo~ ~ thi~ T/C rich aEea o~ ~ codi~g ~eqU~16 CONST~U~ION OF ~0~ DNA 5EQ~OES
S ~LOWI~& ~ PRODIJ~ON OF
Rg~ rE~ 10~ t~ , w~ h~Y~ d~
@~ llo~ g '~ u~ o ~12 ~ i~ 10 ~ ~dI:rI bu~ a~d 100 ,uP~ NaCl ai: 37~C
0 ~. ~2acl~ by ~d~ ~T~
15 (0.25 ~ aDd 1 ~1 r~sor~u~ a~. Aft@~ h~a~g th~ tur~
at 37~t: ~r 5 ~, w~ add~d 1 ~o~ o~ ~h2~0~ d sh~ok W~ uEiied ~L~ ~ized D~ by c~o~ato~:ra~y o~ ~5~ iora~) (@lu~ion with ~0 m~ T~ {Cl (P~ t 1 1~ ~TI~ d pool~d th~ DN~co~tal~g p~a~
(400 ~1). W~ th~ add~d 100 1ll B~l 31~1~ a~d 1 u~t of Bal 31 a~d ~cuba~ 30C o~ d~
t:Lo~ zl~d I:)l!aA, s~ b~ lly a~ le!~3cxib~
by ~ a~ta~ 0~;~@~D~ o~ ~g D~ 5~ ?r~ te~ t i N~
~ 3~, 9~ 1?1?~ 567g-~38 (L981~o W~ took 1010 ~ul aligllo~3 ~xo~ ~ a~ , 20 ~ec, 30 45 ~ ~d ~0 ~ E~ a~ o~ cl~e~
o~ d~l~tio~ ro~ ~ ariz~d DN~, W~ ga~ch~d ~e~ d~ïe~:io~ r~ac~o~ ach ali$uo~
1~ ad~ p~ a~d ~ wi~ ~
ph~ol and o~c~ s~ith C105 ,ul ~ W~2 pr~dpi~t~d ~Ghe ~wi~a~ e~ it~ (8~0 8 ~ Yi~ld- ~10 ~g ~ gach aliquot.
Si~e~ ~18 ~ e~tsiC:l~iO31 5i~ eC~TC~3 i~ pBOEIo 35 iM~t~Ala) i~ locat~d ~ to the ~ t of ~a~ DN~ 3~eqll2~1Ge e~codir~g ~ t 8s~ ~ ~hat plas~d SFi~@ 4), r~ictio~
. ~i~ N~ ~a@~ o~ s@ar~ with ~al 3~
@~a~ u~ e~ e~ g ~13-BGH and therefore will produce DN~ sequences that encode B~H-like polypeptides that lack one or more amino terminal amino acids, as compared to authentic BGH tFigure 4).
In order to select those deletions ending with a T (chosen because of the convenient location of a HindIII
site outside of the BGH coding seguence as described below), we employed the me~hod described by N. Panayotatos and K. Truong, ~ . We dissolved the DNA in 34 ~1 ~2~ added 4 ~1 10 x ~indIII buffer and 20 units ~ind III and incubated the mixture at 37C for 1 h. This restriction reaction cut the non-BGH end of the linearized DN~ at the HindIII
site (Figure 4). We then filled in the HindIII site by mixing the DNA with 5 ~1 1 mM dNTP's, 1 ~1 Klenow (1 unit) and incubated the mixture at 37C for 15 min. After frac-tiona-ting the filled~in DN~ on an agarose gel, we observed two bands: a small 100 base pair band corresponding to the minor HindIII fragment of ~he DNA and a large band corresponding to the major DNA fragmen~ (shown in Figure 4).
We eluted the maior DNA fragment from the gel, precipitated it with EtOH and collected the DNA by centri-fugation ~000 rpm, 8 min). As depicted in Figure 4, this DNA fra~ment carries at one end a filled in HindIII restric-tion site (AAGCT) and at the other end one of the various deletions in the BGH coding seguence produced by the pre-viously dessribed Bal 31 dlgestion. Plainly, recirculari-zation of the DNA will regenerate a HindIII site (AAGCTT) at the point of religation only in those cases where the Bal 31 deletion in the BG~ coding sequence terminates with a T. Therefore, we religated the DNA and cleaved it again with Hin_III to select the desired T-ending deletions (Figure 4).
To recircularize the DNA, we dissolved the EtOH
precipitate from above in 33 ~1 ~2l combined ~hat solution with 4 ~1 10 x ligation salts anA 2 ~1 T4 DNA ligase and incubated the mixture at 14C for 16 h, substantially as described by [N. Panayotatos and K. Truong, supra]. We then stopped the reaction by heating the mixture a~ 70C
for 15 min and isolated the DNA as before.
` . --1 ~ ~ , To isolate the DNA fragments sarrying the desired deletions ~i.e., those terminating in a T), we restricted the recircularized DNA from a~ove with HindIII and BamHI.
As depicted in Figure 4, such restriction allows selection of a DNA fragment containing the deleted ~GH coding sequence.
Accordingly, we dissolved the DNA in 5 ~1 10 x HindIII
huffer, added 20 units H _ III and 20 units BamHI and incubated the mixture at 37~C for 60 min. Af-ter digestion we fractiona~ed ~he mixture on an acrylamide gel and eluted the ~665 base pair fragment of desired DNA from the gel, precipitated it with EtOH and collected it by centrifuga-tion as before. This fragment (in one strand) carries at one end ~he T marking the end of the particular deletion that we effected in ~he B~H coding se~uence with Bal 31 digestion and at the other end the Bam HI cleavage residue which is located beyond the COO~ terminal end of the BGH
coding se~uence. In order to determine the nucleotide sequence of the various deletions (i,e. at which T the deletion stoppPd), we inser~ed our Bam HI-Hind III frag-ment in ind III-Bam HI-linearized pB~322 by dissolving the ~NA in 10 ~1 H20, adding 0.1 ~g ind I~I-Bam HI
digested pB~322 (1 ~1), 1.2 yl 10 x ligation buf~r and O.5 ul T4 DNA ligase and incubated the mixture at 14C
for 16 h.
After stopping the ligation, we transformed 200 ~1 of competent E. coll K12 A ~a gift of Walter Fiers) in the presence of 10 ~1 0.1 ~ MgCl2 by chilling the cells in ice for 15 min, incubating them at 42C for 2 min and incubating them at room temperature for 10 min. After adding 2 ml L-Broth, we grew the cells at 37C for 1 h and plated a 200 ~1 aliquot onto petri pla~Ps, containing L~Broth supplemented with 50 ~g/ml ampicillln. We grew our colonies at 37C for 16 h and randomly selected 24 clones from those colo~ies resistant to ampicillin~
We grew up cultures of each of these clones in 5 ml of L-Broth, supplemented with 50 ~g/ml ampicillin for 12 h at 37C and purified plasmid DNA from 1 ml of cells from each culture by the mi~iprep method described 3~
B~ H~ r~ i~ th~ 24 ~la~mid Di~s by ~d IlI~Bam ~I
ad p~ a~d~ ge~ ~Ea~o~a~A. '~
k~ 3~ D~ ha ng @gu d ~m o~ ola~ R~
aRd ~ployi~y th~ nci~g m~thod d~crib~d by nd Gilb~rt, ~,~ Seq~ci~g~ r~ al~d ~a~ on~ of th~
~t~ ue~d~ 127 tT~ 2 c:ot~a1.~ E~ OE aDI~ e~s ~9 10 ~d th~t ~o~r og ~ r~$ b~aa a~ ~uel~o1~ 112 o:~ ~C~ h~d ~a~e~ d~ t~d;lo ~ S~O~ S~
A~OW~JG ~ P~ODUCT~ON OF
BG~SKEi: :PO~ ?TI~ IN ~ YI~
ow ~ 5~ w~ h~
th~r~ o~of a ~roce~ ~or eo~ rueti~
io~ ~ec:to~ Gh~a6torized ~y a D~ ~que~c~ of thi~
i~Y~ation that allows ~ ~rodu ~io~ o ~ov~ ik~
2û pol~p~d~ i~ high y~ld.
~o att~t to exp~ DN~ ~qu~ ~ tho ~w~ cla~s~ o~ ~or~ d~ d ~ L9 ~, w~
D~ ~3~ f~ la~ ~ b~ ea~
25 Elind I~ r~ Qt wi~ X1Q110W a~ b~for~ d ~h~a cl~avir~g th~ ~ ~i~ 10 ~it~ o B~ ElI und~g ~ co~di~o~
d~scE~b~d ~ ~iou~ly., W~ iod ~ d~ r~d r~lat:~d ~ ~ag~ o~ a 5% ~by w~i~h~ ~oly~e~l~d~
~ Lo Th:~ ~ag~ ~
~e~a~d ~d ~ X~
fi~1~g ~ ~d III ~ a~d th~ ~a1 T ~
~UG~ ~de~ 104 Of F19~ 2 ~9 )O~ aC1~dQ 89 Of E?~ B 2 ~h43 ~, T~ ~r~ OU~ ~S~O~ ~re~ t~ ~:@~ 3 ~3 3 5 ~ W~ a~q~d t~ a~iea1 ~ W~ ~ad p~eviously e~ploy~ to e~r~ BS~ ~equ~c~3 u~d~s th~
co~ol o~ th~ rolaoter (~.g., p~ (Me~t~DAla~) ~ith Nc~ I, fillod i:ll th~ ~a~ wi~ ~ealow a~ el~a~e~d ~ ear^
~16~
,.
i2~dl D~ with B~ El~ u~i~g ~:o~Ye~tio~al eo~ditio~ aDd buff~r$. Thi~ frag~t carrle~ i~ o~ d at o~ 2~1S3 th~ ~e~ned C~TG ~ro~ ~ filli~y i~ of the Nco I ~it~
a~d a~ th~ o~r ~d tho r~idu~ ~o~ Bam ~I clea~a~
S ~Figur~ 53. ~ ~er~ combi~ed 0.2 l~g of th~ prepared ve~tor (2 yl) a~d O.S ~g of th~ p~epæ~d D~ i~ of li~atia~
bu~ d 0.5 ~1 T~ D~ liga~ ~d iIlcubat~d ~m at 1~C
or 16 h.
ixcu~ 2a~:io~l p~:oducQI~ ~0 ~ 0~8, 1~ re~p~cti~ly. I~ C1rl@ ~e ~us:leo~d~ s~qu~c~ be~ g with th~ ATG is A~TT:: f~llow~d by ~o add 6 oi~ ~
BC~ coding ~ c~ of Fis~ 2 ( 2~ ~4 ~ s~r ~ ~ ( Figur~ S ) .
~n ~ o~er th~ ~ucl~o~d~ $e~e~c~ be~q ~i~ th~
ATG i~ GC~ ollowad by a~i~o acid 11 of th~ B~ cod~
15 i~g sQgue~c~ o Figu~ 2 (p~-~9(So~)) (F~ S~ re-fo~, i~ th~ fi~t co~t~uctio~, t~ GOdiIl~ Ee~iO~l fQ:~
the i~ir~t four a~o acid~ o Dla~ BGE~ ha~ b~ r~noYed, th~ codo~ AGC (s~r~e) ix~ort~d a~d ~ codiD~g r~io~ for t~ fifth ~o acid of ~a~ BGEI ~odified ro~ ~TG to 20 ~TG cha~gi~g i~ ~o acid fro~ m~thio~ to leuci~e.
other co~uct:io~ a pr~e~red constructio~ of e$tio~ th@ eo~i~q r~gio~ Xor th~ irst ~o a~o acids o mab~r~ BG8 ha~ be~ ~ red, 1~ eo~o~ e~e~
i~dl a~d th~ coLi~ regio~ ~ th~ te~th ~c~ a::id 25 of ~atur~ odlifi~d rom CT~ ~:o TT~, a ~go that do~3 not a~ct its am~no ~cid (leuci~ r@~l:~t ~h~ f:Lr9t constructio~ should expr~ ~ a pro~ia o ~ ollowi~g ~equ~c~ ~et-S~ r.eu~o acid~ 6-191 of ~a~r@ B~ a~d th~ o'ch~ co~uctio~ ~hould ~pr~$~ a ~otei~ of the 30 follo~ g ~gue~c~ ~e~-ser-~ao aci~10~191 of mature BG8~* Ac~ordi~ly, w~ ha~ de~ignat~d ~ ~o v~ctoæ~
pBY~-~4 ( S~r ~ 9 ( S~x ) ( Figur~ 5 ~ t re~ Cl iYe!ly .
* Of cou~e, i~ should be under~oo~ t~a~ f~t ~5 may be remov~d froDa khe~ poly~epl:ide~ eith2r d~ g eacpres-sio~ or 511b~;el~1@I3.~ her~to to produce polyp~p~ides of ~h~
formllla 5~r-L~u~P~ to A~g~ o B~l and S~r ~loto o ~GEI, re~poc:tiY~ly. rrl ~act, sub~egu~t a~o acid - ~e~u~c~ of BOEl~(S~r) rev~al~d t~a~ f~e~ wa~
. j` 4~ cle~ed ~1~o~ du~i~g o~gpre~sioIl o~ ~Lrifieatio~O
~ 2 We employed the recircularized plasmids to trans-form E. coli K12 A, as descrlbed be~ore, again selecting those colonies that were resis~ant to ampicillin~ We then grew up cultures of 12 of the ampicillin resistant clones, isolated their plasmid DNA ~y the miniprep method, described previously, and si2ed the inserts by Pst I/Eco RI restric-tion using conventional conditions. From the results o~
the sizing, we selec~ed that plasmid DNA having the appro-priate insext. We then used this DNA to transform E. coli MC 1061, substantially as described in G. Nv Buell et al., J. Biol. Chem. 254, pp. 9279-83 ~1979 ) . We also transformed the cells with pcI~57 (a gif~ of W~ Fiers).* We selected cells transformed with our BGH rPlated plasmid ~N~ and pcI~57 by growing khe colonies fox 16 h at 30C in L-Broth, supplemented with ampicillin, as before, and kanamycin ~40 ~g/ml).
We then randomly picked ampicillin and kanamycin-resis~a~t colonies and grew them overnight in 5 ml L-Broth, supplemented as before with ampicillin and kanamycin, at 30C. We diluted the cultures with 25 ml of ~-Broth at 42C and allowed the cells to grow at 42C for 2 h (O.D.=l).*~
To isolate the BGH-like polypeptides produced by the ~ransformed cells, we took 1 ml of cells and spun them down (8000 rpm, 4 min) added 50 ~1 Laemmli buffer ~1% SDS~ [Laemmli, Nature, 2~7, pp. 681 et seg. (1970)], ~oiled the mixture for 15 min, and again centrifuged the c~118 ( ~000 rpm, 4 min) to remove the cellular debris.
We assayed the supernatant by radioimmunoassay for BGH
using rabbit an~isera ~o authentic BGHo ***
* pcI857 is a small plasmid that carries a temperature-sensitive P~ repressor and the gene coding for kanam~cin resistance.
** Thes~ are the identical growth conditions employed previously for P ~controlled expression of BG~ using the DNA sequence cod~n~ for mature BG~.
*** This is -~he identical isolation method and assay pre-viously employed for P -controlled expression of BGH using the DNA se~uence codin~ for mature BGH.
~2 ~ ~ ~3 ~
We obtained the following expression levels of BGH~like polypeptides:
Yield of BGH B~H-like Deletion Activity_(mg/l) Molecules/cell ~4~Ser~ 30.0 1.5 x 106 ~9(Ser) 100 5.0 x 106 We also used the ~4(Ser) and ~9(Ser) DNA sequences in the trp-containing vector previously used to express BGH using the DNA sequence coding for mature BGH. In those constructions, we obtained the following expression levels:
Yield of BGH BGH like Deletion Actlvity (mg~ Molecules/cell ~4(~er) 0.15 8000 ~9(Ser) 0.50 50000 Finally, we manipulated our constructions to delete fxom the constructions the AGC codon (coding for serine) that had been added by the Hind III fill-in. The AGC deleted constructions pBGH-~4 and pBGH-~9 are the more preferred constructions of our invention, with pBGH-a9 ~0 being the most preferred.* To make these constructions, we modified the fill-in of the Hind III site of the BGH-related sequence, described previously, so that only dATP
was present in the mixture. This modification added only a single A during fill-in (Figure 6). We then diluted the reaction mixture 10~fold with Sl ~uf~er ~30 mM NaOAc (pH 4.5), 300 mM NaCl, 3 mM ZnCl2~, added 2 units/~g DNA
of Sl and incubated the mixture at 37C for 30 min to cleave off the unfilled DNA overhang (Fisure 6). We then stopped the reaction with phenol and proceeded as before to restrict the DNA fragmen~ with Bam HI and insert it into the desired expression vector. Using -these vectors, we observed the following expression levels:
* Again the amino terminal f-Met produced on expression of these constructions could be removed from these BGH-related pol~peptides either during expression or subsequent thereto. Because of our experience with the actual amino acid sequence of BGH-~9(Ser), we e~pect -that the f-Met is removed either during expression or purification.
Yield of BGH BGH-like Deletion Promoter Activit~ m~/l Molecules/cell ~9 PL 100 5 x 106 We have also used the ~4 and ag DNA sequences of this inven-tion, as well as a BGH-~l cons-truction prepared by -the methods of this invention, under PL
control (with a mu-derived ribosome binding site, as before) to transform various E. coli hosts in order -to compare expression levels:
Deletion Host~- 12 Yield3 ~1 MC 1061 2 x 104 HB 101 10 x 104 PR 7 30 x 104 a4 MC 1061 3.6 x 105 HB 101 6.0 x 106 PR 7 13 x 105 ~9 MC 1061 1.4 x 106 HB 101 1.1 x 106 PR 7 1.5 x 106 Met-Ala-BGH MC 1061 7.5 ~ 10 (control) PR 7 20 x 102 Therefore, DNA sequences and recombinant DNA molecules characteri~ed by them, having portions of the B~H coding sequence deleted permit the produc-tion of bovine growth hormone-like polypeptides in high yield: at least 100 times higher than DNA
sequences coding for mature bovine growth hormones in the same expression vectors and hosts, and prefer-1 All hosts carried the pcI857 repressor plasmid.
2 Genotypes of the hosts are as follows- R
PR 7 : thr leu ~p thi rns mal xal mtl Sm HB 101 : leu lac pro thi hsr hsm ~e~ recA SmR
MC 1061: ara leu lac ~ hsr hsm StrA
PR 7 : thr leu ~p thi rns mal xal mtl Sm HB 101 : leu lac pro thi hsr hsm ~e~ recA SmR
MC 1061: ara leu lac ~ hsr hsm StrA
3 RIA yields in molecules per cell following induc-tion for 2 h at 42C in shake flasks.
B.0891 ably 1000 times higher. Moreover, such deletions permit the expression of novel bovine growth hormone-like polypeptides, u~eful aft~r purification usin~
conventional methods, as yeneral anabolic agents in S cattle, most especially ~o increase the rate of growth, weight gain and meat production in those animals. E'or example, after p~rification to about 90~ homogeneity, the ~9-BGH produced by our most preferred strain E. coli MC 1061 (PL mu-~9-BGH) dis~
played about 80% of the growth promoting of native BGH in standard rat tibia assays.
The actual extent of ~he amino ~erminal deletion in the DNA sequences of our invention is not cri~ical to our invention and the high yield of the novel BGH-like polypeptides attained by this inven-tion, because the extent of the deletion may depend on ~he expression control sequence or host used. In-stead, the amino terminal deletion should be suffi-cient to permit the DNA sequence to produce at lea~t about 100 times more molecules/cell of a BGH like polypep~ide, and more preferably at least about 1000 times more, than is produced by the DN~ sequence en-coding mature .~GH in a particular host and expression vector. Such DN~ sequences and assays may be con-~tructed by those of skill in the art by following~he teachings and examples of thi~ invention. There-~ore, such DNA sequences, the recombinant DNA mole-cules containing them, and the boYine growth hormone-like polypeptides made ~y them are art of this in~en-~ion.
The actual method employed to afect thedeletions o~ our invention is also ~ot critical. For example, while we have described an embodiment where-in the deletion was effected using Bal 31 and sel-ected by choosing deletions ending ~ith a T, ~.ther deletion methods are ~lso ~seful in our invention.
Such methods include cleaving the BGH coding sequence ~ ~2~
and replacing a part of the cleaved sequence with a synthe-tic fra~ment, Bal 31 digestion and selecting Eor cleletions other than -those ending in T, combina-tions of the -two methods, or other deletion methods known in the ar-t.
It should of course be unders-tood tha-t other hosts and expression vec-tors may also be used to express the novel DNA sequences of ~lis invention and to produce the novel polypeptides of -~his invention in high yield. Such hosts include other strains of E,. coli, as well as strains of Pseudomonas, S-trepto-myces, Bacillus, yeasts and other fungi, and plant and animal cells in cul-ture. ~ is a par-ticularly preferred host. To express the D~ se-quences of this invention in those hosts, the DNA
sequences are inserted into expression vectors com-patible with the particular host selec-ted and those vectors employed as described above.
Microorganisms and recombinant DNA molecules prepared by the processes of this invention are exem-pliied by cultures deposited in the American Type Culture Collection, Rockville, Maryland on Augus-t 16, 1982, an~ identified there as BGH-A to C.
A. E. coli Kl2 ~ (p~GH-(Met-Ala)) B. E. coli Kl2 ~ (pBGH-~4(Ser~) .
C. E. coli Kl2 ~ (p~GH-~9(ser)) These cultures were assigned accession numbers 39173, 39174 and 39175, respec-tively.
While we have hereinbefore presented a num-ber of embodimen-ts of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the processes and compositions of this invention. ~'herefore, it will be appreclated that the scope of this invention is to be de~ined by the claims appended hereto ra-ther than by the specific embodiments which have been presented herelnbefore by way of e~ample.
.
.
B.0891 ably 1000 times higher. Moreover, such deletions permit the expression of novel bovine growth hormone-like polypeptides, u~eful aft~r purification usin~
conventional methods, as yeneral anabolic agents in S cattle, most especially ~o increase the rate of growth, weight gain and meat production in those animals. E'or example, after p~rification to about 90~ homogeneity, the ~9-BGH produced by our most preferred strain E. coli MC 1061 (PL mu-~9-BGH) dis~
played about 80% of the growth promoting of native BGH in standard rat tibia assays.
The actual extent of ~he amino ~erminal deletion in the DNA sequences of our invention is not cri~ical to our invention and the high yield of the novel BGH-like polypeptides attained by this inven-tion, because the extent of the deletion may depend on ~he expression control sequence or host used. In-stead, the amino terminal deletion should be suffi-cient to permit the DNA sequence to produce at lea~t about 100 times more molecules/cell of a BGH like polypep~ide, and more preferably at least about 1000 times more, than is produced by the DN~ sequence en-coding mature .~GH in a particular host and expression vector. Such DN~ sequences and assays may be con-~tructed by those of skill in the art by following~he teachings and examples of thi~ invention. There-~ore, such DNA sequences, the recombinant DNA mole-cules containing them, and the boYine growth hormone-like polypeptides made ~y them are art of this in~en-~ion.
The actual method employed to afect thedeletions o~ our invention is also ~ot critical. For example, while we have described an embodiment where-in the deletion was effected using Bal 31 and sel-ected by choosing deletions ending ~ith a T, ~.ther deletion methods are ~lso ~seful in our invention.
Such methods include cleaving the BGH coding sequence ~ ~2~
and replacing a part of the cleaved sequence with a synthe-tic fra~ment, Bal 31 digestion and selecting Eor cleletions other than -those ending in T, combina-tions of the -two methods, or other deletion methods known in the ar-t.
It should of course be unders-tood tha-t other hosts and expression vec-tors may also be used to express the novel DNA sequences of ~lis invention and to produce the novel polypeptides of -~his invention in high yield. Such hosts include other strains of E,. coli, as well as strains of Pseudomonas, S-trepto-myces, Bacillus, yeasts and other fungi, and plant and animal cells in cul-ture. ~ is a par-ticularly preferred host. To express the D~ se-quences of this invention in those hosts, the DNA
sequences are inserted into expression vectors com-patible with the particular host selec-ted and those vectors employed as described above.
Microorganisms and recombinant DNA molecules prepared by the processes of this invention are exem-pliied by cultures deposited in the American Type Culture Collection, Rockville, Maryland on Augus-t 16, 1982, an~ identified there as BGH-A to C.
A. E. coli Kl2 ~ (p~GH-(Met-Ala)) B. E. coli Kl2 ~ (pBGH-~4(Ser~) .
C. E. coli Kl2 ~ (p~GH-~9(ser)) These cultures were assigned accession numbers 39173, 39174 and 39175, respec-tively.
While we have hereinbefore presented a num-ber of embodimen-ts of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the processes and compositions of this invention. ~'herefore, it will be appreclated that the scope of this invention is to be de~ined by the claims appended hereto ra-ther than by the specific embodiments which have been presented herelnbefore by way of e~ample.
.
.
Claims (9)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS.
1. A process for increasing the yield of a bovine growth hormone-like polypeptide to at least 100 times that of a bovine growth hormone-like poly-peptide encoded by a DNA sequence of the formula:
in the same vector-host expression system, said process comprising the step of culturing a host transformed with a recombinant DNA molecule compris-ing a DNA sequence encoding a Met .DELTA. or .DELTA. bovine growth hormone-like polypeptide operatively linked to an expression control sequence, said .DELTA. being an amino terminal deletion from the amino acid sequence of mature bovine growth hormone.
in the same vector-host expression system, said process comprising the step of culturing a host transformed with a recombinant DNA molecule compris-ing a DNA sequence encoding a Met .DELTA. or .DELTA. bovine growth hormone-like polypeptide operatively linked to an expression control sequence, said .DELTA. being an amino terminal deletion from the amino acid sequence of mature bovine growth hormone.
2. The process of claim 1, characterized in that said DNA sequence encoding the Met .DELTA. or .DELTA.
bovine growth hormone-like polypeptide encodes a polypeptide selected from the group consisting of polypeptides of the formulae: MetPhe-AA3 to AA191 of BGH, Phe-AA3 to AA191 of BGH, MetSerLeu-AA6 to AA191 of BGH, SerLeu-AA6 to AA191 of BGH, MetLeu-AA6 to AA191 of BGH, Leu-AA6 to AA191 of BGH, MetSer-AA10 to AA191 of BGH, Ser-AA10 to AA191 of BGH Met-AA10 to AA191 of BHH and AA10 to AA191 of BGH.
bovine growth hormone-like polypeptide encodes a polypeptide selected from the group consisting of polypeptides of the formulae: MetPhe-AA3 to AA191 of BGH, Phe-AA3 to AA191 of BGH, MetSerLeu-AA6 to AA191 of BGH, SerLeu-AA6 to AA191 of BGH, MetLeu-AA6 to AA191 of BGH, Leu-AA6 to AA191 of BGH, MetSer-AA10 to AA191 of BGH, Ser-AA10 to AA191 of BGH Met-AA10 to AA191 of BHH and AA10 to AA191 of BGH.
3. The process of claim 1, characterized in that said bovine growth hormone-like polypeptide is selected from the group consisting of polypeptides of the formulae: MetPhe-AA3 to AA191 of BGH, Phe-AA3 to AA191 of BGH, MetSerLeu-AA6 to AA191 of BGH, SerLeu-AA6 to AAlgl of BGH, MetLeu-AA6 to AAlgl of BGH, Leu-AA6 to AA191 of BGH, MetSer-AA10 to AA191 of BGH, Ser-AA10 to AA191 of BGH, Met-AA10 to AA191 of BGH and AA10 to AA191 of BGH.
4. The process of any one of claims 1, 2 or 3, characterized in that said DNA sequence encoding the Met .DELTA. or .DELTA. bovine growth hormone-like polypeptide increases the yield of a bovine growth hormone-like polypeptide to at least 1000 times that of a bovine growth hormone-like polypeptide encoded by a DNA
sequence of the formula:
in the same vector-host expression system.
sequence of the formula:
in the same vector-host expression system.
5. A recombinant DNA molecule comprising a DNA sequence encoding a Met .DELTA. or .DELTA. bovine growth hormone-like polypeptide operatively linked to an expression control sequence, said .DELTA. being an amino terminal deletion from the amino acid sequence of mature bovine growth hormone other than a .DELTA.1 dele-tion or a .DELTA.4 deletion and said DNA sequence increas-ing the yield of said Met .DELTA. or .DELTA. bovine growth hormone-like polypeptide to at least 100 times that of a bovine growth hormone-like polypeptide encoded by a DNA sequence of the formula:
in the same vector-host expression system.
in the same vector-host expression system.
6. The recombinant DNA molecule of claim 5, characterized in that the expression control sequence is selected from the group consisting of the lac system, the trp system, the major operator and promoter regions of phage .lambda., the control region of fd coat protein, other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.
7. A bovine growth hormone-like polypep-tide produced by the process of claim 1.
8. A process for producing a bovine growth hormone-like polypeptide comprising the step of culturing a host transformed with a recombinant DNA molecule according to claim 5.
9. A bovine growth hormone-like polypep-tide produced by the process of claim 2, character-ized in that it is selected from the group consisting of polypeptides of the formulae: MetSerLeu-AA6 to AA191 of BGH, SerLeu-AA6 to AA191 of BGH, MetLeu-AA6 to AA191 of BGH, Leu-AA6 to AA191 of BGH, MetSer-AA10 to AA191 of BGH, Ser-AA10 to AA191 of BGH, Met-AA10 to AA191 of BGH and AA10 to AA191 of BGH and other polypeptides coded for by DNA sequences characterized by an amino terminal deletion from the DNA sequence coding for mature bovine growth hormone other than a .DELTA.1 deletion or a .DELTA.4 deletion, said sequences allowing the production of said polypeptide in at least 100 times higher yield than that of a bovine growth hormone-like polypeptide encoded by a DNA sequence of the formula:
in the same vector-host expression system.
in the same vector-host expression system.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8223695 | 1982-08-17 | ||
| GB8223695 | 1982-08-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1224432A true CA1224432A (en) | 1987-07-21 |
Family
ID=10532360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000434118A Expired CA1224432A (en) | 1982-08-17 | 1983-08-08 | Dna sequences, recombinant dna molecules and processes for producing bovine growth hormone-like polypeptides in high yield |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US4693973A (en) |
| EP (1) | EP0103395A3 (en) |
| JP (1) | JPS5963197A (en) |
| AU (1) | AU568597B2 (en) |
| CA (1) | CA1224432A (en) |
| DD (1) | DD212982A5 (en) |
| DK (1) | DK375283A (en) |
| ES (1) | ES524971A0 (en) |
| NO (1) | NO832948L (en) |
| NZ (1) | NZ205212A (en) |
| PT (1) | PT77206B (en) |
| ZA (1) | ZA835880B (en) |
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| US5254463A (en) * | 1981-09-18 | 1993-10-19 | Genentech, Inc. | Method for expression of bovine growth hormone |
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| EP0111814A3 (en) * | 1982-12-16 | 1985-08-14 | Mgi Pharma, Inc. | The cloning and expression of nucleotide sequences encoding bovine growth hormone |
| BG49718A3 (en) * | 1983-07-15 | 1992-01-15 | Bio Technology General Corp | Method for preparing of polypeptid with superoxiddismutasne activitty |
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| DE3585170D1 (en) * | 1984-03-06 | 1992-02-27 | Lilly Co Eli | EXPRESSION VECTORS FOR BOVINE GROWTH HORMON DERIVATIVES. |
| US5489529A (en) * | 1984-07-19 | 1996-02-06 | De Boer; Herman A. | DNA for expression of bovine growth hormone |
| US4861868A (en) * | 1985-02-22 | 1989-08-29 | Monsanto Company | Production of proteins in procaryotes |
| US5141922A (en) * | 1985-02-22 | 1992-08-25 | Monsanto Company | Biologically active proteins and a method for their use |
| US5037806A (en) * | 1985-02-22 | 1991-08-06 | Monsanto Company | Biologically active method using somatotropins |
| US4670249A (en) * | 1985-04-12 | 1987-06-02 | International Minerals & Chemical Corp. | Growth-promoting compositions |
| JPH06104070B2 (en) | 1985-06-26 | 1994-12-21 | ジ・アップジョン・カンパニ− | Purification of somatotropin from transformed microorganisms. |
| US4788144A (en) * | 1985-06-28 | 1988-11-29 | International Minerals & Chemical Corp. | Fermentation process for the high level production of swine growth |
| US4762784A (en) * | 1985-07-15 | 1988-08-09 | International Minerals & Chemical Corp. | Fermemtation process for the high level production of bovine growth hormone |
| US4786501A (en) * | 1985-07-15 | 1988-11-22 | International Minerals & Chemical Corp. | Cylindrical implants for the controlled release of growth hormones |
| AU599922B2 (en) * | 1985-09-10 | 1990-08-02 | Natinco Nv | Improving carcass quality |
| US5631227A (en) * | 1985-09-18 | 1997-05-20 | The Upjohn Company | Somatotropin analogs |
| AU6469886A (en) * | 1985-11-08 | 1987-05-14 | Smithkline Beckman Corporation | Mutant alpha-1-antitryosin and dna sequence therefor |
| US4892764A (en) * | 1985-11-26 | 1990-01-09 | Loctite Corporation | Fiber/resin composites, and method of making the same |
| US5366876A (en) * | 1986-12-31 | 1994-11-22 | Lucky Ltd. | Method for production of bovine growth hormone using a synthetic gene |
| JPH0712318B2 (en) * | 1986-12-31 | 1995-02-15 | 株式会社ラッキー | Method for producing bovine growth hormone using synthetic gene |
| EP0279616A3 (en) * | 1987-02-17 | 1990-03-07 | International Minerals And Chemical Corporation | Swine growth promotion |
| US5240837A (en) * | 1987-02-19 | 1993-08-31 | The Upjohn Company | CDNAS encoding somatotropin, expression vectors and hosts |
| JPH01502640A (en) * | 1987-03-12 | 1989-09-14 | アムジエン・インコーポレーテツド | bovine growth hormone analog |
| US5104806A (en) * | 1987-03-12 | 1992-04-14 | Amgen, Inc. | Porcine growth hormone analogs encoding dna |
| US4888416A (en) * | 1987-03-30 | 1989-12-19 | International Minerals & Chemical Corp. | Method for stabilizing somatotropins |
| US5192669A (en) * | 1987-11-09 | 1993-03-09 | Eli Lilly And Company | Method to produce recombinant proteins using an expression vector comprising transcriptional and translational activating sequences |
| US5063158A (en) * | 1987-11-09 | 1991-11-05 | Eli Lilly And Company | Recombinant DNA expression vector comprising both transcriptional and translational activating sequences |
| US5268284A (en) * | 1988-03-11 | 1993-12-07 | The Upjohn Company | Ribosome binding site |
| WO1990002762A1 (en) | 1988-09-02 | 1990-03-22 | The Rockefeller University | Macrophage-derived inflammatory mediator (mip-2) |
| US5079230A (en) * | 1988-09-12 | 1992-01-07 | Pitman-Moore, Inc. | Stable bioactive somatotropins |
| DK472189A (en) * | 1988-09-29 | 1990-03-30 | Bunge Australia | GROWTH HORMONE |
| WO1990005185A1 (en) * | 1988-11-07 | 1990-05-17 | L'universite De L'etat A Liege | Modified human growth hormone |
| US6787336B1 (en) | 1989-10-12 | 2004-09-07 | Ohio University/Edison Biotechnology Institute | DNA encoding growth hormone antagonists |
| US6583115B1 (en) | 1989-10-12 | 2003-06-24 | Ohio University/Edison Biotechnology Institute | Methods for treating acromegaly and giantism with growth hormone antagonists |
| US5958879A (en) * | 1989-10-12 | 1999-09-28 | Ohio University/Edison Biotechnology Institute | Growth hormone receptor antagonists and methods of reducing growth hormone activity in a mammal |
| US5350836A (en) * | 1989-10-12 | 1994-09-27 | Ohio University | Growth hormone antagonists |
| US5266477A (en) * | 1990-02-02 | 1993-11-30 | Pitman-Moore, Inc. | Monoclonal antibodies which differentiate between native and modified porcine somatotropins |
| ATE196548T1 (en) * | 1991-05-10 | 2000-10-15 | Genentech Inc | SELECTING AGONISTS AND ANTAGONISTS OF LIGANDS |
| GB2273931A (en) * | 1992-09-28 | 1994-07-06 | Unilever Plc | Mutant polypeptides |
| JPH08508640A (en) | 1993-03-10 | 1996-09-17 | スミスクライン・ビーチャム・コーポレイション | Human brain phosphodiesterase |
| SE9303068D0 (en) * | 1993-09-21 | 1993-09-21 | Kabi Pharmacia Ab | New use |
| US5767080A (en) * | 1996-05-01 | 1998-06-16 | Cargill, Incorporated | Enhanced milk production in dairy cattle |
| ZA9711733B (en) | 1996-12-31 | 1998-07-01 | Monsanto Co | Method for solubilization and naturation of somatotropins |
| US6087128A (en) * | 1998-02-12 | 2000-07-11 | Ndsu Research Foundation | DNA encoding an avian E. coli iss |
| US20050256239A1 (en) * | 2004-05-11 | 2005-11-17 | Lee Chun D | White polyolefin compositions having reduced die drool |
| CN107446943A (en) * | 2017-09-12 | 2017-12-08 | 浙江华军药业有限公司 | The expression of bovine growth hormone recombinant |
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|---|---|---|---|---|
| US3265579A (en) * | 1964-01-08 | 1966-08-09 | Upjohn Co | Process for purification of bovine growth hormone |
| US3308027A (en) * | 1965-05-13 | 1967-03-07 | Upjohn Co | Bovine pituitary growth hormone process |
| US3664925A (en) * | 1970-02-20 | 1972-05-23 | Martin Sonenberg | Clinically active bovine growth hormone fraction |
| US4056520A (en) * | 1972-03-31 | 1977-11-01 | Research Corporation | Clinically active bovine growth hormone fraction |
| NZ187300A (en) * | 1977-05-27 | 1982-08-17 | Univ California | Dna transfer vector and micro-organism modified to contain a nucleotide sequence equivalent to the gene of a higher organism |
| IE48385B1 (en) * | 1978-08-11 | 1984-12-26 | Univ California | Synthesis of a eucaryotic protein by a microorganism |
| US4443539A (en) * | 1980-02-05 | 1984-04-17 | The Upjohn Company | Process for preparing bovine growth hormone |
| IL59690A (en) * | 1980-03-24 | 1983-11-30 | Yeda Res & Dev | Production of bovine growth hormone by microorganisms and modified microorganisms adapted to produce it |
| IE52755B1 (en) * | 1980-08-26 | 1988-02-17 | Univ California | Bovine pre-growth and growth hormone |
| JPS57181099A (en) * | 1981-04-29 | 1982-11-08 | Biogen Nv | Bacillus cloning vector, recombinant dna molecule, bacillus host transformed thereby and manufacture of polypeptide expressing dna order and being coded thereby |
| JPS58996A (en) * | 1981-06-01 | 1983-01-06 | ザ・ボ−ド・オブ・トラステイ−ズ・ミシガンステ−トユニバ−シテイ− | Method of cloning ox growth hormone gene in germ |
| NZ201918A (en) * | 1981-09-18 | 1987-04-30 | Genentech Inc | N-terminal methionyl analogues of bovine growth hormone |
| US4371462A (en) * | 1982-01-21 | 1983-02-01 | Genex Corporation | Method for purification of anterior pituitary hormones |
-
1983
- 1983-08-08 CA CA000434118A patent/CA1224432A/en not_active Expired
- 1983-08-08 EP EP83304574A patent/EP0103395A3/en not_active Withdrawn
- 1983-08-10 ZA ZA835880A patent/ZA835880B/en unknown
- 1983-08-11 US US06/522,357 patent/US4693973A/en not_active Expired - Fee Related
- 1983-08-11 NZ NZ205212A patent/NZ205212A/en unknown
- 1983-08-16 DK DK375283A patent/DK375283A/en not_active Application Discontinuation
- 1983-08-16 PT PT77206A patent/PT77206B/en unknown
- 1983-08-16 JP JP58148857A patent/JPS5963197A/en active Pending
- 1983-08-16 ES ES524971A patent/ES524971A0/en active Granted
- 1983-08-16 NO NO832948A patent/NO832948L/en unknown
- 1983-08-16 AU AU18021/83A patent/AU568597B2/en not_active Ceased
- 1983-08-17 DD DD83254030A patent/DD212982A5/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0103395A3 (en) | 1985-05-22 |
| AU568597B2 (en) | 1988-01-07 |
| EP0103395A2 (en) | 1984-03-21 |
| JPS5963197A (en) | 1984-04-10 |
| NO832948L (en) | 1984-02-20 |
| DD212982A5 (en) | 1984-08-29 |
| ES8502476A1 (en) | 1985-01-01 |
| DK375283D0 (en) | 1983-08-16 |
| US4693973A (en) | 1987-09-15 |
| ES524971A0 (en) | 1985-01-01 |
| PT77206B (en) | 1986-06-02 |
| DK375283A (en) | 1984-02-18 |
| AU1802183A (en) | 1984-02-23 |
| NZ205212A (en) | 1987-03-31 |
| PT77206A (en) | 1983-09-01 |
| ZA835880B (en) | 1984-04-25 |
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