Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-11-11
1999-10-12
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
43525421, 530363, 536 231, 536 235, C12N 1500
Patent
active
059653868
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims foreign priority benefits under 35 USC .sctn.119 to International Application Publication No. WO 95/23857, which was filed on Mar. 1, 1995 and which claims priority to GB 94042702, which was filed on Mar. 5, 1994.
FIELD OF THE INVENTION
The present invention relates to the production of recombinant human albumin (rHA) by yeast species.
BACKGROUND OF THE PRIOR ART
Human serum albumin (HSA) is a protein of 585 amino acids that is responsible for a significant proportion of the osmotic pressure of serum, and also functions as a carrier of endogenous and exogenous ligands. It is used clinically in the treatment of patients with severe burns, shock, or blood loss, and at present is produced commercially by extraction from human blood. The production of recombinant human albumin (rHA) in microorganisms has been disclosed in EP 330 451 and EP 361 991.
In recent years yeast species have been widely used as a host organisms for the production of heterologous proteins (reviewed by Romanos et al, 1992), including rHA (Sleep et al, 1990, 1991; Fleer et al, 1991). Yeasts are readily amenable to genetic manipulation, can be grown to high cell density on simple media, and as eukaryotes are suitable for production of secreted as well as cytosolic proteins.
When S. cerevisiae is utilised to produce rHA, the major secreted protein is mature 67 kDa albumin. However, a 45 kDa N-terminal fragment of rHA is also observed (Sleep et al, 1990). A similar fragment is obtained when rHA is expressed in Kluyveromyces sp. (Fleer et al, 1991) and Pichia pastoris (EP 510 693). The fragment has the same N-terminal amino acid sequence as mature rHA, but the carboxy terminus is heterogeneous and occurs between Phe.sup.403 and Val.sup.409 with the most common termini being Leu.sup.407 and Val.sup.409 Geisow et al, 1991), as shown below.
.dwnarw. .dwnarw. -Phe-Gln-Asn-Ala-Leu-Leu-Val-Arg-Tyr-Thr-Lys-Lys-Val-Pro-Gln- (SEQ ID
NO:16)
405 410 415
The amount of fragment produced, as a percentage of total rHA secreted, varies with both the strain and the secretion leader sequence utilised, but is never reduced to zero (Sleep et al, 1990). We have also found that the amount of fragment produced in high cell density fermentation (75-100 g/L cell dry weight) is approximately five times higher than in shake flask cultures.
The 45 kDa albumin fragment is not observed in serum-derived human serum albumin (HSA), and its presence as non-nature-identical material in the recombinant product is undesirable. The problem addressed by the present invention is to reduce the amount of the 45 kDa fragment in the product. The simplest and most obvious approach would have been to have purified it away from the full length albumin, as proposed by Gist-brocades in EP 524 681 (see especially page 4, lines 17-22). However, we have chosen a different approach, namely to try to avoid its production in the first place.
Sleep et al (1990) postulated that rHA fragment is produced within the cell and is not the result of extra-cellular proteolysis. These authors codon-optimised the HSA cDNA from Glu.sup.382 to Ser.sup.419 but this had no effect on production of rHA fragment. They noted that a potential Kex2p processing site in the rHA amino acid sequence, Lys.sup.413 Lys.sup.414, is in close proximity to the heterogeneous carboxy terminus of the fragment, but neither use of a kex2 host strain (ie a strain harboring a mutation in the KEX2 gene such that it does not produce the Kex2p protease), nor removal of the potential cleavage site by site-directed mutagenesis of the codon for Lys.sup.414, resulted in reduction in the amount of the fragment.
There is a vast array of yeast proteases which could, in principle, be degrading a desired protein product, including (in S. cerevisiae) yscA, yscB, yscY, yscS, other vacuolar proteinases, yscD, yscE, yscF (equivalent to kex2p), ysc.alpha., yscIV, yscG, yscH, yscJ, yscE and kex1.
Bourbonnais et al (1991) described an S. cerevisiae endoprotease activity specif
REFERENCES:
patent: 5440018 (1995-08-01), Ohmura et al.
Egel-Mitani et al. (1990) Yeast. A Novel Aspartyl Protease Allowing Kex2-Independent MFalpha Propheromone Processing in Yeast. vol. 6:127-137, 1990.
Bourbonnais et al. (1991) Enzyme. Prohormone Processing by Yeast Proteases. vol. 45:244-256, 1991.
Lawn et al. (1981) Nucleic Acids Res. The Sequence of Human Serum Albumin cDNA and its Expression in E. coli. vol.9, No. 22, pp 6103-6114, 1981.
Kunkel (1985) Proc. Natl. Acad. Sci. USA Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection. vol.82:488-492, 1985.
Barton, "Protein Sequence Alignment and Database Scanning." In Protein Structure Prediction, A Practical Approach, 1996, IRL Press, Oxford University Press at Oxford, UK, pp. 31-63, 1996.
George et al. "Current Methods in Sequence Comparison and Analysis." In Macromolecular Sequencing and Synthesis, Selected Methods and Applications, 1988, D.H. Schlesinger (ed) Alan R. Liss, Inc. New York, NY, pp. 127-149, 1988.
Sleep et al. (1990) Bio/Technology 8:42-46, 1990.
Biochimie, vol. 76, 1994, pp. 226-233, Y. Bourbonnais et al., Cleavage of Prosomatostatins by The Yeast Yap3 and Kex2 Endoprotease.
Biotechnology, vol. 8, No. 1, 1990, pp. 42-46, D. Sleep et al., The Secretion of Human Serum Albumin from The Yeast S. Cerevisiae Using Five Different Leader Sequences.
Embo Journal, vol. 12, No. 1, 1993, pp. 285-294, Y. Bourbonnais et al., Isolation and Characterisation of S. Cerevisiae Mutants Defective in Somatostatin Expression . . .
Yeast, vol. 6, 1990, pp. 127-137, M. Egel-Mitani et al., A Novel Aspartyl Protease Allowing KEX2-Independent MF Alfa Propheromone Processing in Yeast.
Schwartz (1986) FEBS Lett. 200, 1-10.
Brenner et al (1992) Proc. Natl. Acad. Sci. 89, 922-26.
Zhu et al (1992) Mol. Microbiol. 6, 511-20.
Gilbert Sarah Catherine
Kerry-Williams Sean Martin
Biswas Naomi S.
Delta Biotechnology Limited
Longton Enrique D.
Wax Robert A.
LandOfFree
Yeast strains and modified albumins does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Yeast strains and modified albumins, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Yeast strains and modified albumins will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-651145