Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-07-27
1997-03-18
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...
435 698, 4351723, 4352523, 4352542, 4353201, 530363, 530364, 514 8, 514 12, C12N 1509, C12N 1514, C12N 119, C07K 14765
Patent
active
056121962
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a preparation of human serum albumin, a process for producing it, and its uses.
Human serum albumin (HSA) is a non-glycosylated monomeric protein of 585 amino acids, of a molecular weight of 66 KD. Its globular structure is maintained by 17 disulphide bridges which create a sequential series of 9 double loops (Brown, J. R., "Albumin Structure, Function and Uses", Rosenoer, V. M. et al. (eds.) Pergamon Press, Oxford, (1977) 27-51). The genes encoding HSA are known to be highly polymorphic, and more than 30 apparently different genetic variants have been identified by electrophoretic analysis under various conditions (Weitkamp, L. R. et al., Ann. Hum. Genet. 37 (1973) 291-226). The gene for HSA is cut in 15 exons by 14 intron sequences and comprises 16,961 nucleotides, from the supposed "capping" site up to the first site for addition of poly(A).
Human albumin is synthesized in the liver hepatocytes and then secreted in the blood stream. This synthesis leads, in a first instance, to a precursor, prepro-HSA, which contains a signal sequence of 18 amino acids directing the nascent polypeptide in the secretory pathway.
HSA is the most abundant protein in the blood, with a concentration of about 40 g per liter of serum. There are therefore about 160 g of albumin circulating in the human body at any time. The most important role of HSA is to maintain a normal osmolarity of the blood stream. It also has exceptional binding capacity for various substances and plays a role both in the endogenous transport of hydrophobic molecules (such as steroids and bile salts) and in that of various therapeutic substances which can also be transported to their respective sites of action. Furthermore, HSA has recently been implicated in the catabolism of prostaglandins.
HSA represents 40% of the world market for plasma proteins. Its commercial interest lies in the fact that this product is widely used, for example in so called replacement solutions to compensate for blood losses during surgical procedures, accidents or haemorrhages, and at doses which may be as high as several tens of grams per day per individual. Currently, the annual consumption of HSA can be estimated at more than 300 tonnes.
Up until now, the HSA available on the market is produced by purification from biological material of human origin. In particular, it is obtained by conventional techniques for fractionation of plasma obtained from blood donations (Cohn et al., J. Am. Chem. Soc. 68 (1946) 459 pp), or by extraction from human placenta, according to the technique described by J. Liautaud et al. (13th International Congress of IABS, Budapest; A: "Purification of proteins. Development of biological standard", Karger (ed), Bale, 27 (1973) 107 pp).
The development of genetic engineering and new extraction and purification techniques has opened the possibility of obtaining, at a lower cost price, improved products of higher purity, of better stability and without risk of viral contamination (for example hepatitis B and AIDS). Given the importance of the HSA market, the possibility of producing this protein by the recombinant route has been widely studied. Thus, numerous expression systems have been studied for the preparation of recombinant HSA.
More particularly, as regards bacterial hosts, the first genetic engineering experiments used the bacterium E. coli as host organism. Thus, European Patents EP 236 210, EP 200 590, EP 198 745 or EP 1 929 describe processes for the production of HSA in E. coli using various expression vectors, various transcriptional promoters, and various signals for secretion. Subsequently, work relating to the secretion of HSA in Bacillus subtilis was also carried out, even though the levels of albumin obtained in this system still do not appear to be satisfactory (Saunders et al., J. Bacteriol. 1.69 (1987) 2917).
As regards the eucaryotic hosts, processes for the production of HSA have been developed using yeasts as host organism. Thus, it has been possible to demonstrate in S. cervisiae (Etcheverry et
REFERENCES:
patent: 4086222 (1978-04-01), Lindquist et al.
patent: 4097473 (1978-07-01), Lewis et al.
patent: 5330901 (1994-07-01), Prevatt et al.
Becquart J er ome
Fleer Reinhard
Jung G erard
Prouty Rebecca
Rhone-Poulenc Rorer S.A.
Savitzky Martin F.
Smith Julie K.
Wax Robert A.
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