CA1340547C - Novel secretory leader sequences for yeasts - Google Patents

Abstract

Secretory leader sequences, for use in secreting heterologous polypeptides in yeast, are formed by fusing part of the human serum albumin pre-sequence or part of the Kluyveromyces lactis killer toxin pre-sequence to the Saccharomyces cerevisiae mating factor alpha-1 KEX2 cleavage recognition site. The resulting fusion leader sequences are:

(a) H2N-Met-Lys-Trp-Val-Ser-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr-Ser-Arg-Ser-Leu-Asp-Lys-Arg-COOH
or (b) H2N-Met-Asn-Ile-Phe-Tyr-Ile-Phe-Leu-Phe-Leu-Leu-Ser-Phe-Val-Gln-Gly-Ser-Leu-Asp-Lys-Arg-COOH

Conservative variations are also encompassed.

Description

13~05~ 7 Novel Secretory Leader Sequences for Yeasts This invention relates to secretory leader sequences which can be employed to direct the secretion of a heterologous protein (such as human serum albumin) from fungi (for example the yeast Saccharomyces cerevisiae).

Translocation of protein molecules through bi-lipid membranes from one cellular compartment to another generally relies upon information held within the primary amino acid sequence of the protein itself. The most prevalent and therefore the best characterised sequence information is the amino terminal leader or signal sequence of prokaryotic and eukaryotic organisms. Genetic studies in which the signal sequence has been totally or extensively deleted indicate that the signal sequence is essential for protein translocation ~Benson, S.A. et al.
1985, Ann. Rev. Biochem. 54, 101-134). Among several hundred known sequences (Watson, M.~.E., 1984, Nuc. Acid.
Res. 12, 5145-5164) no consensus signal sequence or even an absolute requirement for any amino acid at any given position can be discerned, although a common feature of many leader sequences is a core of 7-10 hydrophobic amino acids. Genetic manipulations which result in alterations to the hydrophobic core, either by deletion or by X

.. . .

13~0547 inserting charged residues, generally result in a block in protein translocation (Benson, S.A., et al. 1985, Ann.
Rev. Biochem. 54, 101-134). Moreover, in a series of extensive modifications to the chicken lysozyme leader sequence, Yamamoto et al. 1987 (Biochem. and Biophys.
Res. Comm. 149, 431-436) have shown that, while some alterations to the hydrophobic core can result in the abolition of secretion, others can potentiate the leader sequence function, resulting in increased levels of protein secretion.

While the leader sequence is usually essential for the translocation of proteins across membranes, once translocated these sequences are usually endoproteo-lytically cleaved by enzymes contained within the cellular compartments into which the proteins have now moved. These enzymes recognise specific amino acid sequences within the primary structure of the translocated protein. Moreover, complete processing of certain eukaryotic proteins to their mature form often relies upon a series of proteolytic cleavages (Bussey, H., 1988 Yeast 4, 17-26).

3 1~0~17 With the recent advances in recombinant DNA
technology, increasing resources have been brought to bear on the commercial exploition of fungi, particularly yeasts, as vehicles for the production of a diverse range of proteins.

Since many of these proteins are themselves naturally secreted products, it is possible to utilise the information contained within the leader sequence to direct the protein through the secretion pathway.
However, this information is contained within a peptide foreign to yeast. Its recognition and subsequent processing by the yeast secretory pathway are not necessarily as efficient as those of a homologous yeast leader sequence. As a consequence an alternative approach has been to replace the leader sequence with one derived from a naturally secreted yeast protein.

The most widely used yeast secretory sequence is the 89 amino acid leader sequence of the alpha-factor mating pheromone. Processing of this leader has been extensively studied (Kurjan & Herskowitz, Cell 30, 933-943, 1982;
Julius et al. 1983 Cell 32, 839-852; Dmochow~ka et al.
Cell 50, 573-584, 1987; Julius et al. Cell 36: 309-318, 1984; Julius et al. Cell 37, 1075-1085, 1984) and 4 13405~7 requires at least four gene products for complete proteolytic cleavage to liberate the mature 13 amino acid alpha-factor pheromone.

Complete proteolytic cleavage of the alpha factor primary translation product requires first the removal of the N-terminal 19 amino acid signal sequence by a signal peptidase within the endoplasmic reticulum. Following this the sequential action of three gene products located within the golgi apparatus processes the large precursor molecule, liberating four copies of the alpha factor pheromone. These are the EEX2 gene product, an endopeptidase which cleaves after the Lys-Arg dibasic amino acid pair, a carboxypeptidase B-like cleavage, recently identified as the product of the E~Xl gene, and a dipeptidyl amino peptidase, the product of the STE13 gene, which sequentially removes the Glu-Ala or Asp-Ala diamino acid pairing preceding the mature alpha-factor pheromone.

The alpha factor prepro leader sequence has successfully been employed to secrete a range of diverse proteins and peptides. However, when the alpha factor signal is used to direct secretion of human serum . , ~ ~ .. ... .

- 1310.~17 albumin, we have found that a large proportion of the extracellular HSA produced is in the form of a 45KD
N-terminal fragment.

EP-A-252 561 tSclavo) discloses the use of the 16 amino acid signal peptide (pre-sequence) from the killer toxin of Kluyveromyces lactis to aid secretion of heterologous proteins in yeast.

A further possibility is to use a fusion secretory leader sequence. This may be generated by the fusion of two independent sequences. A hybrid signal in which the first amino acids of the acid phosphatase signal were fused to the proteolytic cleavage site of human alpha interferon resulted in the expression and secretion of interferon (Hinnen et al. Foundation for Biochemical and Industrial Fermentation ~esearch, 229, 1219-1224, 1983);
10% of the interferon produced was secreted into the medium. In a similar approach the first 22 amino acids of the alpha-factor leader were fused to the last twelve amino acids of the human interferon alpha-2 signal sequence resulting in the secretion of interferon alpha-2 into the culture supernatant (Piggott et al. Curr. Genet.
12 561-567, 1987). An identical construct in which the interferon alpha-2 gene was replaced by the interferon B

6 13~0547 gene did not result in any secretion of human interferon ~ into the culture supernatant. Finally, in a series of experiments designed to assess the effect of leader sequences on the secretion of human lysozyme, Yoshimura et al. (Biochem. & Biophys. Res. Comm. 145, 712-718, 1987) described a fusion leader comprising the first 9 amino acids of the chicken lysozyme leader and the last 9 amino acids of the Asper~illus awamori glycoamylase leader. Although this fusion leader was effective in secreting 60% of the produced material into the culture supernatant, it was only 15% as effective as the entire chicken lysozyme leader. Moreover, no secreted product could be detected if the human lysozyme sequences were preceded by the entire Asper~illus glycoamylaRe leader, or a fusion derived from the first 9 amino acids of the Aspergillus glucoamylase leader and the last 9 amino acids of the chicken lysozyme leader.

We have now devised new and advantageous leader sequences for use in fungi.

One aspect of the invention provides an amino acid sequence as follows:

~a) HzN-Met-Lys-Trp-Val-Ser-Phe-Ile-Ser-Leu-Leu-Phe-Leu-7 13~05'1~

Phe-Ser-Ser-Ala-Tyr-Ser-Arg-Ser-Leu-Asp-Lys-Ar~-COOH
or (b) H2N-Met-Asn-Ile-Phe-Tyr-Ile-Phe-Leu-Phe-Leu-Leu-Ser-Phe-Val-Gln-Gly-Ser-Leu-Asp-Lys-Arg-COOH

or conservatively modified variations of either sequence.

Table 1 shows alternative amino acids for each position except the initial methionine. Any of the possible permutations are within the scope of the invention. The selection of lysine or arginine for the last two positions is particularly non-critical, although there should always be Lys or Arg at each of these positions. Preferably, positions 20 and 21 of sequence (a) are not Gly and Val respectively. Sequences which are up to four amino acids shorter or longer are also included provided that the C-terminal (Lys, Arg), Lys-Lys or Arg-Arg entity is maintained, there is a positively charged residue within 5 residues of the N-terminus and there is a generally hydrophobic region at or adjacent the middle of the sequence.

8 1340~47 Table }

Leader (a) Met Lys Trp Val Ser Phe Ile Ser Leu Leu Phe Leu Phe Ser Arg Phe Leu Thr Trp Leu Thr Ile Ile Trp Ile Trp Thr His Tyr Ile Gly Tyr Val Gly Val Val Tyr Val Tyr Gly Gln Met Ala Met Ala Met Met Met Ala Asn Ser Ala Tyr Ser Arg Ser Leu Asp Lys Arg Thr Thr Phe Thr Lys Thr Ile Glu Arg Lys Gly Gly Trp Gly His Gly Val Asn Ala Ser Ala Gln Ala Met Gln Asn His ; 1340547 Leader (b) Met Asn Ile Phe Tyr Ile Phe Leu Phe Leu Leu Ser Phe Val Asp Leu Trp Phe Leu Trp Ile Trp Ile Ile Thr Trp Leu Glu Val Tyr Trp Val Tyr Val Tyr Val Val Gly Tyr Ile Gln Met Met Met Met Met Ala Met His Gln Gly Ser Leu Asp Lys Arg Asp Ser Thr Ile Asn Arg Lys Asn Thr Gly Val Glu Glu Ala Ala Met Gln His His A second aspect provides a fusion compound comprising any of the said amino acid sequences linked, preferably directly, at the carboxyl terminal to the N-terminal residue of a polypeptide. The polypeptide may be any desired polypeptide, includin~ "pro-polypeptides" (in other words precursors which undergo post-translational cleavage or other modification, such as glycosylation).
The term "polypeptide" encompasses oligopeptides. The polypeptide may be fibronectin or a portion thereof ~for example the collagen or fibrin-binding portions described in EP 207 751 by DeltaBiotechnology Limited published Jan 7/87), urokinase, pro-urokinase, the 1-368 ..~

- 1340.5q7 portion of CD4 (D. Smith et al (1987) Science 328, 1704-1707), platelet derived growth factor (Collins et al (1985) Nature 316, 748-750), transforming growth factor (Derynck et aI (1985) Nature 316, 701-705), the 1-272 portion of Von Willebrand's Factor (Bontham et al, Nucl.
Acids Res. 14 7125-7127), the Cathepsin D fragment of fibronectin (585-1578), ~ l-antitrypsin, plasminogen activator inhibitors, factor VIII, ~-globin, ~-globin, myoglobin or nerve growth factor or a conservative variant of any of these. The polypeptide may also be a fusion of HSA or an N-terminal portion thereof and any other polypeptide, such as those listed above.
Preferably, the polypeptide is a naturally-occurring human serum albumin, a modified human serum albumin or a fra~ment of either, such modified forms and fragments being termed "variants". These variants include all forms or fragments of HSA which fulfill at least one of the physiological functions of HSA and which are sufficiently similar to HSA, in terms of structure (particularly tertiary structure) as to be regarded by the skilled man as forms or fragments of HSA.

In particular variants or fragments of HSA which retain at least 50% of its ligand-binding properties, for example with respect to bilirubin or fatty acids, 1340~47 (preferably 80%, or 95%) are encompassed. Such properties are discussed in Brown, J.R. & Shockley, P.
11982) in Lipid-Protein Interactions 1, 26-68, Ed. Jost, P.C. & Griffith, O.H.

The portion of HSA disclosed in EP 322 094 by Delta Biotechnology Limited is an example of a useful fragrnent of HSA which may be secreted by use of the leader sequences of the invention.

A third aspect provides a nucleotide sequence coding for any of the said amino acid sequences or for the said fusion compound. The nucleotide sequence (or the portion thereof encoding the leader sequence) may be selected from the possibilities shown in Tables 2 & 3, for sequences (a) and Ib) respectively, where the codons encoding each amino acid are listed under the amino acids. The codons of Tables 2 and 3 clearly relate to RNA, but it is to be understood that equivalent DNA
nucleotide sequences are also within the scope of this aspect of the invention.

12 1340.5~ 7 Table 2 Met Lys Trp Val Ser Phe Ile Ser Leu Leu Phe Leu Phe Ser AUG AAA UG& GUU UCU UUU AUU UCU UUA UUA UUU WA UUU UCU
AAG GUC UCC UUC AUC UCC UUG UUG UUC UUG UUC UCC
GUA UCA AUA UCA CUU CUU CW UCA
GUG UCG UCG CUC CUC CUC UCG
AGU AGU CUA CUA CUA AGU
AGC AGC CUG CUG CUG AGC

Ser Ala Tyr Ser Ar~ Ser Leu Asp Ly~ Arg UCU GCU UAU UCU CGU UCU UUA GAU AAA CGU
UCC GCC UAC UCC CGC UCC UUG GAC AAG CGC
UCA GCA UCA CGA UCA CUU CGA
UCG GCG UCG CGG UCG CUC CGG
AGU AGU AGA AGU CUA AGA
AGC AGC AGG AGC CUG AGG

13 105~7 Table 3 Met Asn Ile Phe Tyr Ile Phe Leu Phe Leu Leu Ser Phe Val AUG AAU AUU UUU UAU AUU UUU UUA UUU UUA UUA UCU UUU GUU
AAC AUC UUC UAC AUC UUC UUG WC UUG UUG UCC W C GUC
AUA AUA CUU CW CUU UCA GUA
CUC CUC CUC UCG GUG
CUA CUA CUA AGU
CUG CUG CUG AGC

Gln Gly Ser Leu Asp Lys Arg CAA GGU UCU UUA GAU AAA CGU
CAG GGC UCC UUG GAC AAG CGC
GGA UCA CUU CGA
GGG UCG CUC CGG
AGU CUA AGA
AGC CUG AGG

A fourth aspect provides a DNA construct comprising a suitable control region or regions and a nucleotide sequence as defined above, the sequence being under the control of the control region. By "suitable control region" we mean such DNA regions as are necessary to enable the said nucleotide sequence to be expressed in 13~05~7 the host for which the construct is intended. The control region will usually include transcriptional start and stop sequences, 3'-polyadenylation sequences, a promoter and often, an upstream activiation site for the promoter. The man skilled in the art will readily be able to select and assemble suitable regions from those available in this art. However, specific examples of suitable expression vectors and their construction include those disclosed in EP 198 745 by Genetica published October 22, 1986; GB 2 171 703 ~for B. subtilis) by Agency of Industrial Science and Technology (Japan~ published September 3, 1986; EP 207 165 by Earth Chemical Co. published July 3, 1986; EP 116 201 by Chiron published August 22, 1984;
EP 123 544 by Genentech published October 31, 1984; EP 147 198 by Delta Biotechnology Limited published July 3, 1985;
EP 201 239 by Delta Biotechnology Limited published November 12, 1986; EP 248 637 by Delta Biotechnology Limited published December 9, 1987; EP 251 744 by Delta Biotechnology Limited published January 7, 1988; EP 258 067 by Delta Biotechnology Limited published March 2, 1988; EP 286 424 by Delta Biotechnology Limited published October 12, 1988 and EP 322 094 by Delta Biotechnology Limited published June 28, 1989.
A fifth aspect provides a host transformed with the said DNA construct. The host may be any host in which the construct is found to work adequately, including bacteria, yeasts, filamentous fungi, insect cells, plant cells and ~nir~l cells. Preferably, however, the host is Saccharomyces cerevisiae or Schizosaccharomyces pombe, most preferably the former. As many native secretion signals are effective in heterologous hosts (for example the natural HSA leader sequence in yeast) it is entirely reasonable to suppose that the leader sequence of the invention will function in hosts other than yeasts.

1340~ 17 A sixth aspect provides a process for preparing a polypeptide, comprising cultivating the said host and obtaining therefrom the polypeptide expressed by the said nucleotide sequence, or a modified version thereof.

By "modified version thereof", we mean that the actual polypeptide which is separated may have been post-translationally modified, in particular by cleavage of the leader sequence.

A seventh aspect provides a polypeptide prepared by such a process.

So that the invention may be more readily understood, preferred aspects will now be illustrated by way of example and with reference to the accompanying drawings in which:

Figure 1 is a restriction map of plasmid p~113;

Figure 2 is a restriction map of plasmid pEK25;

Figure 3 is a restriction map of plasmid pAYE230;

Figure 4 is a restriction map of plasmid pAYE238;

16 i3~g~q 7 Fi~ure 5 i-~ a restriction map of plasmid pAY~305;

Fi~ure 6 is a restriction map of plasmid pAYE305i Figure 7 is a nucleotide sequence coding for a fusion compound of the invention; and Figure 8 is a nucleotide sequence coding for a fusion compound of the invention.

~xample of a prior art tYPe of leader sequence The DNA coding sequence for mature HSA protein haq been p~aced immediately downstream of a DNA sequence encoding the KEX2 cleavage site of the alpha factor pre pro leader sequence (85 amino acids). When this protein sequence i8 placed under the control of a promoter on a yeast autonomously replicating plasmid and transformed into a haploid strain of the yeast Saccharomyces cerevisiae, mature HSA can be detected in the culture supernatant. N-terminal amino acid sequence information indicates that the secreted protein ha4 the same N-terminal amino acid composition as natural HSA, namely Asp-Ala-His. This also indicate~ that the first two amino acids of the secreted HSA are not susceptible to the dipeptidyl endopeptidase, the product of the STE13 gene, as this enzyme is responsible for the removal of such sequences from between successive repeats of the alpha-factor pheromone. Although mature HSA is the major ~, .

~ . ~

17 13 10~7 product observed in the culture supernatant, a N-terminal fragment of HSA (45 kilodaltons~ was also detected, representing approximately 15X of the total HSA
synthesised. This fragment component represents not only a waste of secretion capacity but also certain downstream purification problems in that, as a fragment of HSA, it shares some biochemical and biophysical properties with intact HSA.

.. . . . . .... .

We have constructed a fusion leader which may be regarded as the natural HSA leader sequence from which the last five amino acids have been removed, to be replaced by the five amino acids preceding the KEX2 cleavage site of the alpha-factor pre pro leader sequence, i.e. amino acids 81 to 85 are Ser-Leu-Asp-Lys-Arg ~Table 2).

When transformed with suitable plasmid vectors incorporating the fusion leader, yeast secrete mature HSA
into the culture supernatant at levels comparable to that observed with the alpha-factor leader sequence. N-terminal sequence analysis indicates that the mature HSA
possesses the correct N-terminal amino acid composition.

Moreover, substitution of the alpha-factor leader by the fusion leader sequence has been found to result in a 6 fold reduction in the levels of the 45 kd fragment observed in the culture supernatant. This therefore represents a significant improvement in the reduction of the contaminating polypeptides, thus aiding the purification of mature HSA from yeast culture supernatants.

1340~j 17 Details Unless otherwise stated all procedures were carried out as described by Maniatis et al ~1982). Plasmid pEK113 (Figure 1) ~EP-A-248 637) was digested to completion with the restriction endonucleases MstII and HindIII. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. The linearised plasmid DNA was then treated with the Klenow fragment of E.coli DNA polymerase I to generate a linearised DNA molecule with blunt ends.

The following oligonucleotide duplex (I) was constructed on an automated Applied Biosystems Inc 380B
DNA synthesiser (according to manufacturer's instructions).

Oligonucleotide I

5, 3' GGC TTA TAA GGA TCC TTA TAA GCC
CCG AAT ATT CCT AGG AAT ATT CG&

The oligonucleotide duplex was ligated with equimolar quantities of linearised, blunt ended p~K113.
E.coli strain ~C1061 was transformed with the ligation 1340~7 mixture and cells receiving DNA were selected on an ampicillin-containing medium (50ug/ml ampicillin).
Recombinant plasmids containing the oligonucleotide duplex were screened by digesting DNA prepared from individual colonies with the restriction endonucleases MstII and EcoRI. Plasmid pEK25 was thus formed tFigure 2~.

Plasmid pEK25 was digested to completion with the restriction endonucleases XbaI and BamHI, DNA fragments were separated by electrophoresi~ through a lX (w/v) agarose gel and a 688 base pair XbaI - BamHI DNA fragment recovered from the gel by electroelution.

The plasmid mpl9.7 ~EP-A-248 637) was digested to completion with the restriction endonuclease XhoI. The linearised DNA was phenol/chloroform extracted and ethanol precipitated. The recovered DNA was then treated with the Klenow fragment of E. coli DNA polymerase I as previously described, following which the DNA was phenol/
chloroform extracted and ethanol precipitated. The recovered DNA was then digested to completion with XbaI

and the digestion products separated by agarose gel electrophoresis. A 1067 base pair fragment was recovered 1~405~17 from the gel by electroelution. The following oligo-nucleotide duplex ~II) was prepared as described prevlously .

Oligonucleotide II

GATCC ATG AAG TGG GTA AGC TTT ATT TCC CTT CTT TTT CTC
TAC TTC ACC CAT TCG AAA TAA AGG GAA GAA AAA GAG

TTT AGC TCG GCT TAT TCC AGG AGC TTG GAT AAA AGA
AAA TCG AGC CGA ATA AGG TCC TCG AAC CTA TTT TCT

22 1340~4 7 The plasmid pUC19 (Yanisch-Perron et al. 1985) was dige~ted to completion with the restriction endonuclease BamHI. Linearised ~NA was recovered by phenol/chloroform extraction and ethanol precipitation.

Equimolar quantities of the BamHI digested pUC19, the oligonucleotide duplex II, the 1067 b.p. DNA fragment derived from mpl9.7 and the 688 b.p. DNA fragment derived from pEK25 were ligated together. E.coli DH5 was trans~
formed with the ligated DNA and transformants selected on 50ug/ml ampicillin L-broth agar. Recombinant colonies containing the desired plasmid, designated pAYE 230 (Figure 3) were selected by digested DNA obtained from individual colonies with the restriction endonuclease Bam~I.

Plasmid pAYE 230 was digested to completion with BamHI and the products separated by electrophoresis through a 1% agarose gel. The 1832 base pair fragment containing the HSA coding sequence was recovered by electroelution.

.. ,.. ~ ...

23 134~7 Plasmid pMA91 (Mellor et al. 1983) was digested to completion with B lII under stsndard conditions. The linearised plasmid was phenol/chloroform extracted and ethanol precipitated.

Equivalent quantities of the linearised pMA91 and the DNA fragment prepared from pAYE 230 were ligated under standard conditions. E. coli DH5 was transformed with the ligation mixture and cells receiving the DNA
selected on L-broth agar containing 50ug/ml ampicillin.
Colonies containing the desired plasmid, designating pAYE
238 (Figure 4) were selected by digesting the DNA from such colonies with PvuII.

Plasmid pAYE 238 was transformed into the yeast Saccharomyces cerevisiae strain S150-2B as described by Hinnen et al. (1978). Cells receiving plasmid pAYE 238 were selected on minimal medium, supplemented with 2X
(w/v) glucose, 20mg/l histidine, 20mg/l tryptophan and 20mg/l uracil.

Transformed S150-2B cells were transferred to lOml YEPD media containing 2% ~w/v) glucose and incubated at 300C, 200rpm for 72 hours. Cell free culture supernatants were analysed by discontinuous native 8-25X gradient polyacrylamide gel electrophoresis on a Pharmacia Phast System, as described in the manufacturer's instructions.
Cells were stained and destained and the relative quantities of native HSA and HSA fragment estimated by gel scan at 595nm.

We have also constructed a second fusion leader which consists of the 16 amino acid pre region of the 97,000 dalton Kluyveromyces lactis killer ~ORF 2) toxin (Stark and Boyd, 1986, Tokumaga et al 1987) fused to the five amino acids preceding the KEX2 cleavage site of the alpha-factor prepro leader sequence, i.e. amino acids 81 to 85, Ser-Leu-Asp-Lys-Arg (Table 3).

When transformed with plasmid vectors incorporating the fusion leader described in Table 3, yeast secreted mature HSA into the culture supernatants at levels higher than when either the natural K.lactis prepro killer toxin leader sequence or the alpha-factor prepro leader sequence was used. N-terminal sequence analysis indicates that the mature HSA possesses the correct N-terminal amino acid composition.

... ... ., . ~ .

13~0547 Substitution of the alpha-factor leader by the K.lactis killer/alpha factor fusion leader sequence resulted in a six fold reduction in the levels of the 45kd fragment observed in the culture supernatant. This therefore represents a significant improvement in the reduction of the contaminating polypeptides, thus aiding the purification of mature HSA from yeast culture supernatants.

Details The experimental procedures employed to generate a yeast HSA secretion vector utilising the K.lactis killer/alpha factor fusion leader were identical to those described in Example 1, except that oligonucleotide duplex (II~ was replaced by oligonucleotide duplex (III) synthesised on an automated Applied Biosystems Inc. 380B DNA synthesiser ~according to manufacturer's instructions).

26 13~0547 Oligonucleotide duplex III

GATCC ATG AAT ATA TTT TAC ATA TTT TTG TTT TTG CTG TCA TTC
TAC TTA TAT AAA ATG TAT AAA AAC AAA AAC GAC AGT AAG

GTT CAA GGA AGC TTG GAT AAA AGA
CAA GTT CCT TCG AAC CTA TTT TCT

Equimolar quantities of the BamHI digested pUC19, the oligonucleotide duplex III, the 1067bp DNA fragment derived from mpl9.7 and the 688b.p. DNA fragment derived from pEK25 were ligated together. ~.coli DH5 was transformed with ligated DNA and transformants selected on 50ug/ml ampicillin B-broth agar. Recombinant colonies containing the desired plasmid, designated pAYE304 (Figure 5), were selected by digested DNA obtained from individual colonies with the restriction endonuclease BamHI.

Plasmid pAY~304 was digested to completion with BamHI and the products separated by electrophoresis through a 1%
agarose gel. The 1823 base pair fragment containing the HSA coding sequence was recovered by electroelution.

134~547 Pla4mid pMA91 (Mellor et al, 1983) was digested to completion with BglII under standard conditions. The linearised plasmid was phenol/chloroform extracted and ethanol precipitated.

Equivalent quantities of the linearised pMA91 and the DNA
fragment prepared from pAYE304 were ligated under standard conditions. E.coli DH5 was transformed with the ligation mixture and cells receiving DNA selected on L-broth agar containing 50ug/ml ampicillin. Colonies containing the desired plasmid, designating pAYE305 ~Figure 6), were selected by digesting the DNA from such colonies with PvuII.

Plasmid pAY~305 was transformed into the yeast Saccharomyces cerevisiae strain S150-2B as described by Hinnen et al, (1978). Cells receiving plasmid pAYE305 were selected on minimal medium, supplemented with 2%
(w/v) glucose, 20mgtl histidine, 20mg/1 tryptophan and 20mg/1 uracil.

Transformed S150-2B cells were transferred to 10ml YEPD
medium containing 2% (w/v) glucose and incubated at 300C, 200rpm for 72 hours. Cell free culture supernatants were ... . . . . . ,.. .~ . .

28 13~0s 17 analYsed by discontinuous native 8-25% gradient polyacrylamide gel electrophoresis on a Pharmacia Phast System, as described in the manufacturer's instructions.

Cells were stained and destained and the relative quantities of native HSA and HSA fragment estimated by gel scan at 595nm.

Using a vector based on the disintegration vectors of EP286424 (Delta Biotechnology), a suitable promoter and the fusion leader of Example 1 above, Schizosaccharomyces pombe (strain Leul.32h was transformed and fermented at 30~C in 10ml of EMM (Edinburgh minimal medium, Ogden, J.E. & Fantes, P.A. (1g86) Curr. Genetics 10 509-514), buffered to pH 5.6 with 0.lM citric acid/sodium phosphate, to give 10-15 mg~l of HSA in the culture supernatant after 3 days.

29 1340~7 References Beggs, J. D. (1978) Nature 275, 104-109.

Beggs, J. D. (1981), Molecular Genetics in Yeast, Alfred Benzon Symp. 16, 383-395.

Birnboim, H. C. & Doly, J. tl979) Nucl. Acids Res. 7, 1513-1523.

Hanahan, D. (1983) J. Mol. Biol. 166, 557-580.

Henderson, R. C. A., Cox, B. S. & Tubb, R. ~1985) Curr.
Genet. 9, 135-136.

Hinnen et al. (1988) PNAS 75: 1929.

Hitzeman, R. A., Clarke, L. & Carbon, J. (1980) J. Biol.
Chem., 255(24), 12073-12080.

Hitzeman, R. A., Hagie, F. E., Levine, H. L., Goeddel, D.
V., Ammerer, G. & Hall, B. D. (1981~ Nature 293, 717-722.

13~05~7 Julius, D., Brake, A., Blair, L., Kunisawa, R. & Thorner, J. (1984) Cell 37, 1075-1089.

Maniatis et al. (lg82~ Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.

Mandel, M. & Higa, A. (1970) J. Mol. Biol. 53, 159-162.

Mellor et al. (1983) Gene 24, 1-14.

Sherman, F., Fink, G. R. & Lawrence, C.(1979) Methods in Yeast Genetics, Cold Spring Harbor, N.Y.

Sleep, D., Belfield, G. P. & Goodey, A. R. (1988) Yeast 4 (14th Int. Conf. on Yeast Genet. & Mol. Biol., Helsinki, Conf. Poster).

Stark, M. J. R. & Boyd, A. (1986) E.M.B.O.J. 5, lg95-2002.

Tokumaga, M., Wada, N. & Hishinuma, F. l1987) Biochem.
Biophys. Res. Comm. 144, 613-619.

Towbin, H., Staehelin, T. & Gordon, J. (1979). P.N.A.S.
76, 4350-4354.

Vogel~tein, B. (1987), Anal. Biochem. 160, 115-118.

Yanisch-Perron et al. (1985) Gene 33 103-109.

Claims (19)

1. A polypeptide having an amino acid sequence as follows:

(a) Met Lys Trp Val Ser Phe Ile Ser Leu Leu Phe Leu Phe Ser Arg Phe Leu Thr Trp Leu Thr Ile Ile Trp Ile Trp Thr His Tyr Ile Gly Tyr Val Gly Val Val Tyr Val Tyr Gly Gln Met Ala Met Ala Met Met Met Ala Asn Ser Ala Tyr Ser Arg Ser Leu Asp Lys Arg Thr Thr Phe Thr Lys Thr Ile Glu Arg Lys Gly Gly Trp Gly His Gly Val Asn Ala Ser Ala Gln Ala Met Gln Asn His or (b) Met Asn ILe Phe Tyr Ile Phe Leu Phe Leu Leu Ser Phe Val Asp Leu Trp Phe Leu Trp Ile Trp Ile Ile Thr Trp Leu Glu Val Tyr Trp Val Tyr Val Tyr Val Val Gly Tyr Ile Gln Met Met Met Met Met Ala Met His Gln Gly Ser Leu Asp Lys Arg Asp Ser Thr Ile Asn Arg Lys Asn Thr Gly Val Glu Glu Ala Ala Met Gln His His wherein vertically aligned groups of amino acids represent alternative amino acids at the position show, and positions 20 and 21 of sequences (a) are not Gly and Val respectively, or a polypeptide which is up to four amino acids shorter or longer, provided (i) that C-terminal Lys-Arg, Arg-Lys, Lys-Lys or Arg-Arg entity is maintained, (ii) that there is a positively charged residue within 5 residues of the N-terminus, (iii) that there is a generally hydrophobic region at or adjacent the middle of the sequence.

and (iv) that the three amino acids N-terminal to the C-terminal Lys/Arg, Lys/Arg entity are maintained.

2. A polypeptide according to claim 1 having an amino acid sequence as follows:

(a) Met Lys Trp Val Ser Phe Ile Ser Leu Leu Phe Leu Phe Ser Arg Phe Leu Thr Trp Leu Thr Ile Ile Trp Ile Trp Thr His Tyr Ile Gly Tyr Val Gly Val Val Tyr Val Tyr Gly Gln Met Ala Met Ala Met Met Met Ala Asn Ser Ala Tyr Ser Arg Ser Leu Asp Lys Arg Thr Thr Phe Thr Lys Thr Ile Glu Arg Lys Gly Gly Trp Gly His Gly Val Asn Ala Ser Ala Gln Ala Met Gln Asn His or (b) Met Asn Ile Phe Tyr Ile Phe Leu Phe Leu Leu Ser Phe Val Asp Leu Trp Phe Leu Trp Ile Trp Ile Ile Thr Trp Leu Glu Val Tyr Trp Val Tyr Val Tyr Val Val Gly Tyr Ile Gln Met Met Met Met Met Ala Met His Gln Gly Ser Leu Asp Lys Arg Asp Ser Thr Ile Asn Arg Lys Asn Thr Gly Val Glu Glu Ala Ala Met Gln His His wherein vertically aligned groups of amino acids represent alternative amino acids at the position show, or a polypeptide which is up to four amino acids longer provided that positions 20 and 21 of sequence (a) and not Gly and Val respectively.

3. A polypeptide according to claim 1 or 2 and having an amino acid sequence:
(a) H2N-Met-Lys-Trp-Val-Ser-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr-Ser-Arg-Ser-Leu-Asp-Lys-Arg-COOH
or (b) H2N-Met-Asn-Ile-Phe-Tyr-Ile-Phe-Leu-Phe-Leu-Leu-Ser-Phe-Val-Gln-Gly-Ser-Leu-Asp-Lys-Arg-COOH.

4. A fusion compound comprising a polypeptide according to Claim 1, 2 or 3 linked at the carboxyl terminal to the N-terminal residue of a second polypeptide.

5. A fusion compound according to Claim 4 wherein the polypeptide of Claim 1 or 2 is linked directly to said second polypeptide.

6. A fusion compound according to Claim 5 wherein the second polypeptide is a naturally-occurring human serum albumin (HSA) a modified human serum albumin having a tertiary structure similar to that of HSA and having at least one physiological function in common with HSA, or a fragment of either.

7. A polynucleotide sequence encoding a polypeptide according to Claim 1, 2, or 3 or a fusion compound according to claim 4.

8. A polynucleotide according to Claim 7 selected from the sequences shown in Figures 7 and 8.

9. A DNA construct comprising a suitable control region or regions and polynucleotide according to Claim 7 or 8, said polynucleotide being under the control of the control region.

10. A host transformed with a DNA construct according to Claim 9.

11. Saccharomyces cerevisiae or Schizosaccharomyces pombe according to Claim 10.

12. A process for preparing a polypeptide, comprising cultivating a host according to Claim 10 or 11 and obtaining therefrom the polypeptide expressed by the said polynucleotide or a post-translationally modified polypeptide thereof.

13. The process as claimed in claim 12, wherein the polypeptide is human serum albumin

14. A polypeptide obtained by a process comprising the following steps:
cultivating a fungal host transformed with a DNA construct, said construct including a suitable control region or regions, a nucleotide sequence coding for said polypeptide and a nucleotide sequence coding for the amino acid sequence of claim 1, said sequences being under the control of the control region, whereby said host expresses and secretes said polypeptide; and recovering said polypeptide.

15. A polypeptide obtained by a process comprising the following steps:
cultivating a fungal host transformed with a DNA construct, said construct including a suitable control region or regions, a nucleotide sequence coding for said polypeptide and a nucleotide sequence coding for the amino acid sequence of claim 1, said nucleotide sequence being selected from the sequences shown in Figures 7 or 8, said sequences being under the control of the control region, whereby said host expresses and secretes said polypeptide; and recovering said polypeptide.

16. The polypeptide of claim 14, wherein said polypeptide is human serum albumin.

17. The polypeptide of claim 15, wherein said polypeptide is human serum albumin.

18. The use of the host as claimed in claim 10 or 11 to prepare a polypeptide.

19. The use of the host as claimed in claim 10 or 11 to prepare human serum albumin.