FI104255B - Process for Preparation of Fusion Proteins Containing N-Terminal Fragments of Human Serum Albumin - Google Patents

Description

104255

The present invention relates to a process for the preparation of fusion polypeptides, wherein two separate polypeptides, or portions thereof, are fused to form a single amino acid chain. Such fusion may result from the expression of a single continuous coding sequence generated by recombinant DNA techniques.

Fusion polypeptides are known, e.g. those in which the polypeptide, which is the final product of the process, is expressed in the "leader" of the N-terminal, which promotes or enables secretion of the polypeptide from the cell. EP-A-116201 discloses an example.

Human serum albumin HS A (human serum albumin) is a known blood protein. EP-A-147198 discloses its expression in a transformed host, in this case yeast. Our previous patent application EP-A-3 222 094, in turn, discloses fragments of the N-terminal HSA, more particularly residues 1-n, where n is 369-419, which can be used therapeutically. The application also mentions the possibility of fusing the C-terminal residue of such molecules to other, unnamed polypeptides.

The present invention relates to a process for the preparation of a fusion polypeptide comprising, at least a portion of the N-terminus, an N-terminal portion of HSA or a variant thereof, and, at least a portion of the C-terminus, a second polypeptide, except that · * ... the N-terminal portion of HSA is 1-n, where n is 369-419 or a variant thereof, the said polypeptide is · · ·

• · I

• · · I

• *:: a) human fibronectin component 585-1578 or a variant thereof; · ·· v: 30 b) CD4 component 1-368 or a variant thereof; c) platelet-derived growth factor or a variant thereof: T: d) transforming growth factor or its variant, 'j'; (e) Human mature plasma fibronectin part 1 -261 or a variant thereof; (f) Human mature plasma fibronectin part 278-578 or variant thereof; (g) Human mature von Willebrandt factor part 1-272 or a variant thereof; or * ·; · 1 h) alpha-1 antitrypsin or a variant thereof.

The essential features of the invention are set forth in the appended claims.

2 104255 The N-terminal of HSA should preferably be said part 1-n, part 1-177 (up to cysteine including cysteine), part 1-200 (up to cysteine excluding cysteine) or intermediate part 1-177 and 1-200 .

The term "human serum albumin" (HSA) refers to, but is not limited to, known or not yet found polymorphic forms of HSA. For example, Naskapi albumin has Lys-372 instead of Glu-372 and Christchurch's pro-albumin has a different pro sequence. The term "variant" refers to, but is not limited to, minor artificial changes in sequences (e.g., 10 molecules lacking one or more residues with conservative substitutions or small residue additions, or having minor changes in the amino acid structure). Polypeptides having 80%, preferably 85%, 90%, 95% or 99% homology to HSA are considered "variants". It would also be desirable that such variants be physiologically equivalent to HSA, i.e. that the variants have at least one pharmacological use of HSA. In addition, the putative variants intended for pharmacological use should not be immunogenic in the target organism (in particular in humans).

Conservative substitutions are those in which one or more amino acids are replaced by others with similar properties that at least the secondary structure, preferably the tertiary structure, of the polypeptide can be expected by a person skilled in the chemistry of polypeptides. Some typical such substitutions are the replacement of glutamine with asparagine, the replacement of asparagine with serine, and the replacement of lysine with arginine. The variants may alternatively or equally lack up to ten (preferably only one or two) intermediate amino acid sequences (i.e., which are not located in the said N-terminus of the HSA terminal moiety) compared to the corresponding moiety in natural HSA. Preferably · *:: such interruptions could be located in the parts of the molecule 100-369 (relative to mature: HSA) (if any). Likewise, at most ten, preferably one or two 30 or two amino acids may be added, further to portion 100-369 (if any).

The term "physiologically functional equivalents" also includes larger molecules containing this said sequence, as well as an additional sequence at the N-terminus (e.g., pro-HS A, pre-pro-HS A and met-HS A).

'; '' 35 It is clear that said "other polypeptides" cannot be the remainder of the HSA, '· ..: otherwise the entire polypeptide would be HSA which would not then be the "fusion polypeptide: T".

• · · • · • · 3 104255

Even when the HSA-like moiety is not referred to as HSA moiety 1-n, it is preferred that the non-HSA moiety is one of said alternatives a) to h).

Part 1-368 of CD4 covers the first four disulfide-bridged immunoglobulin-like domains of the human T lymphocyte protein CD4. Its gene and amino acid sequence are disclosed in D. Smith et al., (1987) Science 328, 1704-1707. It is used against HIV infections.

Human platelet growth factor is described by Collins et al., (1985) Nature 316, 748-750. The sequences of transforming growth factor β (TGF-β) are described by Derynck et al.

(1985) Nature 316, 701-705. These growth factors are suitable for use in wound healing.

The cDNA sequence of Fn part 1-261 is disclosed in EP-A-207751 (obtained from plasmid 15 by the pvu II endonuclease pFH6). This moiety binds fibrin and can be used to direct fused compounds to blood clots.

R.J. Owens and F.E.Baralle, 1986, EMBO J 5, 2825-2830 disclose the 278-578 cDNA sequence for Fn. This part binds to platelets.

Von Willebrandt factor 1-272 binds and stabilizes factor VIII. The sequence is described in Bontham et al., Nucl. Acids Res. 14, 7125-7127.

. . , Variants of alpha-1 antitrypsin are described by Rosenburg et al. 1984, Nature 312, 77-8080. In particular, the present invention includes a Pittsburgh variant (Met is mutated to arginine) and a variant in which Pro357 and Met358 are mutated to one of alanine and the other to arginine. These compounds can be used to treat septic shock and lung diseases.

The variants of the non-HSA moieties of the polypeptides described above include those discussed above in connection with the HSA moiety, including those having conserved amino acid substitutions as well as homologues from other species.

«* <« · · • li

Fusion polypeptides may have amino acids at the N-terminus extending beyond the portion of the N-terminal portion of HSA. For example, if the HSA-like portion is the N-terminal portion of the mature · · · · · HSA, pre-, pro-, or pre-pro-sequence: ': *: for example, the yeast alpha factor leader sequence may be added. The fused conductors shown in WO-90/01063 may be used. The polypeptide to be fused to the HSA moiety 4,104,255 may be a naturally occurring polypeptide, fragment or novel polypeptide, including a fusion polypeptide. For example, in Example 3, the fibronectin fragment is attached to the HSA moiety by four amino acid linkers.

It has been found that the amino terminal portion of the HSA molecule is structured to favor particularly efficient translation and transport of the fusion compounds of the invention in eukaryotic cells.

Another object of the invention is a transformed host having a nucleotide sequence in the order that it expresses a fusion polypeptide as described above. By "such order" we mean, for example, that the nucleotide sequence is in the correct reading sequence with respect to the appropriate RNA polymerase binding site and translation initiation sequence and under the control of a suitable promoter. The promoter may be homologous or heterologous to the host. Downstream (3 ') control sequences may be included as is known in the art. The host should preferably be yeast (e.g. Saccharomyces spp. E.g. S. cerevisiae, Kluyveromyces spp. E.g. K. lactis, Pichia spp. Or Schizosaccharomyces spp. E.g. S. pombe) but may be any other suitable host. , e.g., E. coli, B. subtilis, Aspergillus niger spp., a mammalian cell, a plant cell, or an insect cell.

20

The object of the invention is therefore a method for preparing the described fusion polypeptide by culturing a transformed host according to another aspect of the invention and subsequently isolating the fusion polypeptide in a useful form.

In particular, the methods of the invention are intended to enhance the secretion of human proteins suitable for therapeutic use from yeast, and are invented. These proteins are normally fused only inefficiently to the amino terminal moieties of HSA. One such protein is a potentially valuable wound healing polypeptide having amino acids 585-1578 of human ··· v: 30 fibronectin (hereinafter referred to as Fn 585-1578). As we have described in a separate (appended) appendix, this molecule has cell division, chemotactic, and chemokinetic effects that promote: *; , wound healing. Fusion polypeptides having the C-terminal moiety Fn585-1578,. . when biosynthesized, can be used to heal wounds, especially when a human protein 35 hybrid is used externally. However, amino acid portion 585- '' 1578 of human fibronectin may, if desired, be recovered from the fusion protein by inserting, before the first 'i' of the fibronectin moiety, amino acids having an X-factor cleavage site. After isolation of the fusion protein from the culture supernatant, the desired molecule is liberated using X-factor cleavage and purified by a suitable chromatographic method (e.g., ion exchange chromatography). Other enzymatic or chemical cleavage site sites can be used, either by juxtaposing suitable N-terminal and C-terminal moieties or by inserting 5 suitable linkers between them.

At least some of the fusion polypeptides described, particularly those with said CD4 and vWF fragments, PDGF and ali AT, have an extended half-life in the blood and are therefore advantageous and therapeutically useful: the non-HSA-10 portion of the molecule is therapeutically useful. In the case of ajAT and others, the compound is usually administered in a single dose or in a few small doses over a short period of time, whereby the compound is less likely to induce an immune response.

The examples use standard recombinant DNA techniques as described by Maniatis et al. (1982 and recent 2nd edition) unless otherwise noted. The techniques described in Messing (1983) and Sanger et al (1977) were used to construct and analyze phage-M13 recombinant clones.

The DNA sequences encoding human serum albumin used in the assembly of the molecules were derived from mHOB12 and pDBD2 (EP-A-322094) or by synthesizing oligonucleotides corresponding to portions of this sequence. DNA sequences encoding portions of human fibronectin were derived from pFHDEL1 or .v. oligonucleotides corresponding to portions of this sequence were synthesized. Plasmid '25 pFHDEL1, containing the entire cDNA encoding human plasma fibronectin, was obtained by ligation of DNA derived from pFH6, 16, 54,; ··: * 154 and 1 (EP-A- 207 751).

This DNA is a mRNA variant that does not contain the 'ED' sequence and has 89- • · v; 30 amino acid variation of the CS-CS region (R.J. Owens, A.R. Komblihtt and F.E. Baralle, 1986, Oxford Surveys on Eukaryotic Genes 3, 141-160). The map of this vector: T: is attached (Fig. 11) and the protein sequence of the mature polypeptide produced by *; ', expression of this cDNA, is shown in Fig. 5.

Oligonucleotides were synthesized using an oligonucleotide synthesizer (Applied Biosys tems 3 80 B) according to the manufacturer's instructions.

• «· tl · t · • lit I · I · • t« 6 104255

An expression vector was prepared in which the DNA encoding the HSA secretion signal and mature HS Aita at amino acid 387, including leucine, fused to the same number of DNAs encoding a human fibronectin segment from amino acid 585 to 1578 inclusive was inserted , which was a highly efficient galactose inducible promoter (according to EP-A-258067) which is active in Saccharomyces cerevisiae. Immediately after the 1578 amino acid codon of human fibronectin came a stop codon (TAA) followed by a gene transcription terminator of S. cerevisiae phosphoglycerase kinase (PGK). This vector was then introduced by S. cerevisiae transfor-10 moiety and directed expression and hybridization of a hybrid molecule corresponding to 387 amino acids of the N-terminal HSA fused to amino acids 585-1578 of human fibronectin.

The invention is illustrated by the following examples, in which said figures: Figure 1 (on two sides) depicts the amino acid sequence currently believed to best represent natural HSAs, boxed with alternative C-terminals of HSA (In), Figure 2 (on two sides) the sequence encoding mature HSAs, underlined in linker 3, Figure 3 is a diagram illustrating the structure of mHOB 16, Figure 4 is a diagram illustrating the structure of pHOB31, Figure 5 illustrates (on page 6) the mature protin sequence encoded by the Fn plasmid. pFHDELl,!. Figure 25 schematically illustrates the structure of pDBDF5, Figure 9 schematically illustrates the construction of pDBDF9, Figure 8 schematically illustrates the construction of pDBDF5, Figure 9 schematically illustrates the construction of pDBDF9, Figure 9 schematically illustrates the construction of pDBDF9, construction of plasmid pDBDF12, plasmid v: 30 midiapFHDEL1 is used, and Figure 11 is a map of plasmid pFHDEL1.

··· • · · · · ·

Example 1 HSA 1-387 Fused to Fn 585-1578 v: 35 «« ·

The following describes how to construct plasmids having sequences which: * · *; encode a portion of HSA (EP-A-322094).

• · • · · 7 104255

The human serum albumin coding sequence used to assemble the following molecules was derived from M13mpl9.7 (EP-A-201239) or by synthesizing oligonucleotides corresponding to portions of this sequence. Oligonucleotides were synthesized using phosphoramidite chemistry and an olinucleotide synthesizer (Applied 5 Biosystems 380B) according to the manufacturer's instructions.

An oligonucleotide (linker A), representing part of the known HSDA coding sequence (Figure 2), was synthesized from PstI (1235-1240, Figure 2) to the 381 codon, the codon changed from GTG to GTC.

10

Linker 1

D P H E C Y 5 'GAT CCT CAT GAA TGC TAT

15 3'ACGT CTA GGA GTA CTT ACG ATA

1247

The KVFDEFK

GCC AAA GTG TTC GAT GAA TTT AAA

20 CGG TTT CAC AAG CTA CTT AAA TTT

1267

P LV

CTT GTC 3 '• «4 i.' 25 GGA CAG 5 '•« 1 · Linker 1 was ligated to vector M13mpl9 (Norrander et al., 1983) digested with PstI and HincIL. and the ligation mixture was used to transfect E. coli strain XL1-Blue (Stratagene Cloning Systems, San Diego, CA). Recombinant clones identify • »· ...

v: 30 was determined that they did not turn blue in a medium containing the chromogenic indicator X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) IPTG (isopropylthio-β-galactoside) acid. DNA sequence analysis using template DNA made from bacteriophage particles of a recombinant clone identified a molecule having the required DNA sequence and was designated: 35 mHOB12 (Figure 3).

• M

• · • · • · · · · II.

104255 M13mpl9.7 consists of the coding region for mature HSA in M13mpl9 (Norrander et al., 1983) so that the codon for the first HSA amino acid, GAT, covers the only Xhol site: 5 Asp Ala 5 'CT CGAGAT GC A3'

3 'GAGCTCTACGT51 XhoI

10 (Ep-A-210239). M13mpl9.7 was digested with XhoI, the ends smoothed by S1 nuclease treatment, and then ligated to the following oligonucleotide (linker 2):

Linker 2 15 5 'T CTTT T AT CCAAGCTT GGAT A A A A G A 3' 3 'AGAAAAT AGGTTCGAACCT ATTTTCT 5'

Hind III

The ligation mixture was used to transfect E. coli XL 1-Blue, template DNA was prepared from multiple plaques and then DNA sequence analysis was performed to identify the correct sequence clone pDBD1 (Figure 3).

A 1.1 kb fragment was isolated from pDBD1 by agarose gel electrophoresis

HindIII-Pst-I corresponding to the 5-terminus of the HSA coding region plus half of the inserted 25 oligonucleotide linker. This fragment was then ligated into double stranded: mi1 'mHOB12 pre-digested with HindIII and PstI, and the ligation mixture was then used to transfect E. coli XL1-Blue. Single-stranded template DNA was prepared from mature plaque bacteriophage particles of multiple plaques. The DNA was made double-stranded by in vitro extraction from a heat-treated (annealed) sequence of v1-130 chimeras using a Klenow fragment of DNA polymerase I in the presence of deoxynucleoside triphosphates. Restriction enzyme analysis of this DNA was able to · 1 · 1: identify a clone with the correct configuration, mHOB 15 (Figure 3).

«« <• «4 • The following oligonucleotide (linker 3) covers the region of the mature HSA 382nd amino- '1' 35 acid codon (glutamate, GAA) to lysine codon 389, followed by the termination :: stop codon (TAA). , Hindlll and then the BamHI cohesive head: «1« ·

Linkker 3 9 104255

E E P Q N L I K J

5 'GAA GAG CCT CAG AAT TTA ATC AAA TAA GCTTG 3' 5 3 'CTT CTC GGA GTC TAT TTT ATT CGAACCTAG 5' This was ligated into double-stranded mHOB15 pre-digested with Hind and BamHI. Following ligation, the DNA was digested with Hindi to destroy all non-recombinant molecules and then used to transfect E. coli XL1-10 Blue. The single-stranded DNA was prepared from bacteriophage particles of several clones and subjected to DNA sequence analysis. One of the clones having the correct DNA sequence was designated mHPB16 (Figure 3).

A molecule was created in which the mature HSA coding region was fused to the HSA secretion signal by insertion of linker 4 into BamHL and XhoL digested M13mpl9.7 to give pDBD2 (Figure 4):

Linker 4

20 M K W V S F

5 'GATCC ATG AAG TGG GTA AGC TTT

G TAC TTC ACC CAT TGC AAA

I S L L F L F S

i.25 ATT TCC CTT CTT TTT CTC TTT AGC

\ .y TAA AGG GAA GAA AAA GAG AAA TCG

♦ · • · ··· ♦ • ♦

: S A Y S R G V F

TCG GCT TAT TCC AGG GGT GTG TTT

: J: 30 AGC CGA ATA AGG TCC CCA CAC AAA

: T: R R

. · *: ·. CG 3 'GCAGCT 5' * '··': 35 «i« In this linker, after the parent methionine, the codon for the fourth amino acid and the threonine ACC (Lawn et al., 1981) have been exchanged for serine AGC,. '1 *. resulting in a Hindm site.

• «104255 10

Synthetic DNA sequence corresponding to part of a known HSA coding sequence (Lawn et al., 1981) (amino acids 382-387, Fig. 2) fused to a known fibronectin coding sequence (Komblihtt et al., 1985) (amino acids 585- , Figure 2), was prepared by synthesizing six oligonucleotides (Linker 5, Figure 5). Oligonucleotides 2, 3, 4, 6, 7, and 8 were phosphorylated using T4 polynucleotide kinase, and then oligonucleotides were annealed to 1 + 8, 2 + 7, 3 + 6, and 4 + 5 under standard conditions. The ligated oligonucleotides were mixed and ligated with mHOB 12, which had been digested with restriction enzymes Hindi and EcoRI. The ligation mixture was then used to transfect E. coli XL Ι-ΙΟ Blue. Single stranded template DNA was prepared from mature plaque bacterial phage particles of multiple plaques and DNA sequence analysis was performed. The clone in which the linker of the desired sequence was correctly inserted in the vector was called pDBDFL (Figure 7). This plasmid was then digested with PstL and EcoRI and the approximately 0.24 kb fragment was purified and ligated with the 1.29 kb fragment of pDBD2 in BamHI-PstI (Figure 15 7) and pUC19 digested with BamHI + EcoRI. (Yanisch-Perron et al., 1985) and pDBDF2 was obtained (Figure 7).

A plasmid containing the DNA sequence encoding human fibronectin along its entire length, pFHDEL1, was digested with EcoRL and XhoI, isolated with a 0.77 kb EcoRI-XhoI fragment (Figure 8), then ligated to EcoRI and SalI-digested M13mpl8. (Norrander et al., 1983) and pDBDF3 was obtained (Figure 8).

The following oligonucleotide linker (linker 6) according to EP-A-207751 was synthesized. . from the PstI site 4784-4791 of the fibronectin sequence to the tyrosine codon 1578 (Fig. 5), I, 25 followed by the stop codon (TAA), the HindIII site, and then the BamHI cohesive end: Linker 6 • «

: T: 30 GPDQTEMTI EGL

GGT CCA GAT CAA ACA GAAATG ACT ATT GAAGGCTTG A CGT CCA GGT CT A GTT TGT CTT TAC TGA TAA CTT CCG AAC

• · · • · ·;: 7 Q P T V E Y Stop

: 35 CAG CCC ACA GTG GAG TAT TAA GCTTG

C .: GTC GGG TGT CAC CTC AT A ATT CGAACCTAG

104255 This linker was ligated into PstI and HindIII digested pDBDF3 and obtained pDBDF4 (Figure 8). The following DNA fragments were then ligated together with BglII-digested pKV50 (EP-A-258067) as shown in Figure 8: 0.68 kb EcoRI-BamHI fragment of pDBDF4, 1.5 kb of pDBDF2: n the BamHI-StuI fragment and the 2.2 kb StuI-EcoRI fragment of pFHDEL1. The resulting plasmid pDBDF5 (Fig. 8) contains a promoter according to EP-A-258067 to direct expression of a HSA secretion signal fused to DNA encoding mature HSA amino acids 1-387, which in turn is fused directly and for the same sequence to human fibronect5 amino acid. -1578 encoding DNA, translation terminates with the ket-10 jun stop codon TAA. It is then followed by a S. cerevisiae PKG gene transcriptional modulator. This plasmid also contains sequences that allow selection and maintenance in Escerichia coli and S. cerevisiae (EP-A-258067).

The plasmid was introduced into S. cerevisiae S150-2B (leu2-3 leu2-112 ura3-52 trpl289 his3-1) by standard methods (Beggs, 1978).

The transformants were analyzed and found to produce HSA-fibronectin fusion protein.

Example 2 HSA 1-195 Fused to Fn 585-1578 In this second example, the first region of human serum albumin (1-195),. fused to amino acids 585-1578 of human fibronectin.

· '25 ♦ «

Plasmid pDBD2 was digested with restriction enzymes BamHI and BglII, and the 0.79 kb * ·· ♦ * fragment was purified and ligated into BamHI digested M13mpl9 to give: pDBDF6 (Figure 9). The oligonucleotide • 30 '5'-C CAAAGCTCGAGGAACTTCG-3' was used as a mutagenic primer to create an XhoI site in pDBDF6, in vitro mutagenesis (kit for Amersham Intemation1 PLC). The point was created by changing the HSA \ base number 696 from T to G (Figure 2). The plasmid thus formed was designated '35 pDBDF7 (Figure 9). The following linker was then synthesized to correspond to this newly created XhoI site to HSA lysine codon 195 (AAA) and then to fibronectin: * · *; 585 to the ends of oligonucleotides 1 and 8 of Figure 6.

• · · • «• ♦ • ♦ ♦ 12 104255

Linker 7

D ELRDEGKAS S AK

TC GAT GAA CTT CGG GAT GAA GGG AAG GCT TCG TCT GCC AAA

5 A CTT GAA GCC CT A CTT CCC TTC CGA AGC AGA CGG TTT

I TETPSQPNH

ATC ACT GAG ACT CCG AGT CAG C

TAG TGA CTC TGA GGC TC A GTC GGG TTG AGG GTG G

This linker was then ligated to the heat-treated oligonucleotides 2 + 7, 3 + 6 and 4 + 5 shown in Figure 3 together with XhoI and EcoRI digested pDBDF7 (Figure 9). Note that inserting linkkerm 7 and other oligo-15 nucleotides into pDBDF7 to rebuild the original HSA DNA sequence, and thus the amino acid sequence, does not re-create the XhoI site.

The 0.83 kb BamHI-StuI fragment of pDBDF8 was purified and then ligated with the 0.68 kb EcoRI-BamHI fragment of pDBDF2 and the 2.22 kb Stul-EcoRI fragment of PFHDEL1 in BglII. digested pKV50 and pDBDF9 was obtained (Figure 20 9). This plasmid is otherwise similar to pDBDF5 but specifies only residues 1-195 of HSA, residues 1-387 of pDBDF5.

When introduced into C. cerevisiae S150-2B described above, this plasmid directs. expression and secretion in a hybrid molecule formed by residues 1-5,195 of HSA attached to fibronectin residues 585-1578.

♦ · • ♦ · ···

Example 3: HSA 1-387 Fused to Fn 585-1578 Cleavable Molecule To produce large amounts of fibronectin residues 585-1578, a structure was prepared in which the DNA encoding HSA residues 1-387 was separated from fibronectin residues 585-1578 of the coding DNA with the sequence • · · 13 104255 which specifies the cleavage recognition site by blood coagulation factor X. It follows that the purified secreted product can be treated with factor X and then the fibronectin moiety of the molecule can be separated from the HSA moiety.

For this purpose, two oligonucleotides were synthesized, heat-treated and a linker 8 was obtained.

Linker 8

10EEPQNLI EG GAA GAG CCT CAG AAT TTA ATT GAA GGT

CTT CTC GGA GTC TTA AAT TAA CTT CCA

Rl TETPSQP 15 AGA ATC ACT GAG ACT CCG AGT CAG C

TCT TAG TGA CTC TGA GGC TCA GTC GGG

N S H

TTG AGG GTG G

This linker was ligated with the heat-treated oligonucleotides 2 + 7, 3 + 6 and 4 + 5 shown in Figure 6 into HindIII and EcoRI digested mHOB12 and obtained pDBDF10 (Figure 10). This plasmid was digested with PstI and Eco RI, the approximately 0.24 kb fragment was purified and ligated together with the 1.29 kb BamHI-1 of pDBD2. '25 with the PstI fragment and BamHI and EcoRI digested pUC19 and pDBDF17 was obtained (Figure 10).

The 1.5 kb BamHI-StuI fragment of pDBDF11 was then ligated to 0.68 kb of pDBDF4

With the EcoR1-BamHI fragment and the 2.22 kb StuI-EcoRI fragment of pFHDEL1: T: 30 in BglII digested pKV50 and pDBDF12 was obtained (Figure 10). This plasmid was introduced into S. cerevisiae S150-2B. The purified secreted fusion protein was treated with factor X to release the fibronectin fragments corresponding to the residues 585-1578 of the original molecule.

• · «« «•« «* *« «•« «• I ·« · · · · «« · •

Claims (5)

104255 A method for making a fusion polypeptide comprising, at least a portion of the N-terminus, an N-terminal portion of HSA or a variant thereof, and, at least a portion of a C-terminal portion, a second polypeptide, except that said N-terminal portion of HSA is 1- n wherein n is 369-419 or a variant thereof, said polypeptide is a) human fibronectin part 585-1578 or a variant thereof, b) CD4 part 1-368 or a variant thereof, c) platelet growth factor or a variant thereof, d) transforming growth factor β or a variant thereof, e) human mature plasma fibronectin moiety Ι-ΙΟ 261 or a variant thereof, f) human mature plasma fibronectin moiety 278-578 or a variant thereof; h) an alpha-1 antitrypsin or a variant thereof, wherein the transformed host cell expresses a polynucleotide sequence encoding said fusion polypeptide. 15 Method according to claim 1, characterized in that the fusion polypeptide further comprises at least one N-terminal amino acid extending over the portion corresponding to the N-terminal portion of HSA. Method according to claim 1 or 2, characterized in that there is a cleavable region at the junction of the N-terminal and C-terminal portions of the fusion polypeptide. A method according to any one of the preceding claims, characterized in that the C-terminal portion of the filament polypeptide is a portion of human plasma fibronectin 585-1578 <25: or a variant thereof. • i • I • · * • · « 5. A polynucleotide sequence having a nucleotide sequence in such a sequence; wherein it expresses the fusion poly *: ** peptide as claimed in any one of the preceding claims. : T: 30 • M t · «• · · i t 1 • ·« i 4 · • «· iit • t« • «1 • ·« 4 9 4 I I «f i i lt · 15 104255