Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1995-05-25
1998-07-07
Marshcel, Ardin H.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
435 691, 4351723, 530300, 530333, 530350, C12P 2100
Patent
active
057767246
ABSTRACT:
The mechanisms and components required for chaperonin-dependent folding of proteins has been elucidated using the groEL and groES proteins of E. coli to reconstitute enzymatic activity of two monomeric enzymes, dihydrofolate reductase (DHFR) and rhodanese following dilution from denaturant. The essential elements for properly folding any protein are Mg-ATP (provided in the preferred embodiment as 5 mM Mg acetate and 1 mM ATP), groEL or hsp60, and groES or eukaryotic equivalent. These can be provided in purified form or as a semi-purified cell extract. The groES eukaryotic equivalent, encoded by a gene which does not hybridize to the groES gene, can be isolated using the same technique as was described to isolate Hsp60: isolation of a temperature sensitive lethal yeast mutant (petite at permissive temperature) defective in folding and assembly of imported proteolytically processed human ornithine transcarbamylase (OTC). The yeast mutant is used to identify a yeast genomic DNA sequence that rescues the mutant following library transformation. The rescuing DNA is isolated, characterized, and expressed. The expressed protein is used to make an antibody which is in turn used to identify the protein in yeast mitochondrial extracts and facilitate biochemical isolation of the protein.
REFERENCES:
Goloubinoff et al. (1989) Nature, vol. 342, pp. 884-888.
Lubben et al. (1990) Proc. Natl Acad. Sci (USA), vol. 87, No. 19, pp. 7683-7687.
Hartl Franz-Ulrich
Horwich Arthur L.
Marshcel Ardin H.
Yale University
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