Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-11-21
1998-10-06
Prouty, Rebecca E.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351721, 43525233, 43525421, 4353201, C12P 2106, C12N 1500, C12N 120, C12N 116
Patent
active
058174842
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to an .alpha.-1-antitrypsin (hereinafter referred as "AT") mutein with an enhanced thermoresistance and a process for the preparation thereof.
More particularly, the present invention relates to an AT mutein having an enhanced thermoresistance while maintaining its activity wherein at least one amino acid of a wild-type AT is replaced with another amino acid residue, a polynucleotide encoding said AT, a vector comprising said polynucleotide, a microorganism transformed with said vector, and a process for the preparation of AT with enhanced thermoresistance using said microorganism.
DESCRIPTION OF THE PRIOR ART
The stability of a protein is essential to maintaining its function since it determines the lifetime in vivo and storage time of the protein.
Accordingly, it is desirable for a therapeutic agent or diagnostic reagent comprising proteins to have an improved stability for the commercial practice thereof.
Since the conventional proteinous therapeutic agents isolated and purified from human body have such problems as the limitation of sources and the contamination by various infectious agents, e.g., AIDS or hepatitis virus, there have been many attempts made to produce the therapeutic agents by employing a recombinant DNA technology. However, because the proteins produced by employing the technology generally have a lower stability compared to those isolated from human body, their half-lifetime tends to be reduced markedly when administered into human body. To overcome such stability problem, two approches have been studied: one is to produce a fully glycosylated protein as in its natural form, based on the fact that the decrease in the stability is due to lack or insufficiency of the glycosylation for the protein in a microorganism; and the other is to produce a recombinant protein with an increased stability but maintaining the activity by modifying the amino acid sequence of the protein.
In this connection, it is known that the thermoresistance of a protein is closely related to its stability against denaturation (see Pace, Trends in Biotechnology, 8, 93-98 (1990)).
On the other hand, AT is synthesized in liver cells and then secreted into blood, and classified into serpin family together with many inhibitors of serine proteases such as trypsin, chymotrypsin, elastase, collagenase, thrombin or plasmin. AT is a glycoprotein having a molecular weight of 52 KD and physiologically serves as an inhibitor of elastase in neutrophil. In particular, it protects elastic fibers present in alveoli pulmonis from the degradation by the neutrophil elastase.
Various genetic deficiencies with regard to one's ability to produce AT are well known (see Carrell et al., Mol. Biol. Med., 6, 35-42 (1982)). owing to the genetic deficiencies, the concentration of AT in blood plasma is reduced to break down a balance between a protease and its inhibitors, whereby lung loses its elasticity and there may occur emphysema (Gadek and Crystal, in Metabolic Basis of Inherited Disease, Stanbury et al., Eds., McGraw-Hill, N.Y., pp. 1450-1467). Further, emphysema may result from an inactivation of AT due to an excessive smoking or severe environmental pollution.
For the treatment of these disorders, therefore, the demand for AT has been increasing on a large scale; and AT isolated from human blood has been unable to meet the demand. Also, AT may be used for the treatment of acute shock syndrome (Robin W. Carrell, Biotechnology and GeneticEngineering Reviews, 4, 291-297 (1986)). The shock syndrome is known to be caused by the breakdown of a balance between plasma serpins and proteases due to a sudden massive release of neutrophil elastase.
The nucleotide sequence of a gene encoding AT has already been known (Long et al., Biochemistry, 23, 4828 (1984)); and, the AT gene has been cloned and expressed in Escherichia coli (Bollen et al., FEBS Lett., 166, 67 (1984); Courtney et al., Proc. Natl. Acad. Sci. USA., 81, 669 (1984); Tessier et al., FEBS Lett., 208, 183 (1986); Johnsen et al.,
REFERENCES:
Patston et al. (1990) Biol. Chem. 265, 10786-10791.
Kwon Ki-Sun
Lee Kee-Nyung
Shin Hwa-Soo
Yu Myeong-Hee
Korea Green Cross Corporation
Korea Institute of Science and Technology
Prouty Rebecca E.
Slobodyansky Elizabeth
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