PURPOSE: To obtain a DNA capable of coding a protein, containing a specific amino acid sequence and having human-derived glyoxalase I activity, and producing a human-derived glyoxalase I useful for enzymically synthesizing antiinflammatory agents, screening, etc., carcinostatic agents.
CONSTITUTION: A human lymphoma cell strain V-937 (ATCC CRL1593) is cultured in a culture medium containing 5% bovine fetal blood serum at 37°C in a 5% CO2, gas stream and subsequently treated with phorbol myristate acetate for 16hr. An mRNA is then isolated from the resultant cell according to a conventional method and used as a template to synthesize a cDNA, which is then treated with a restriction enzyme, ligated to a vector and transduced into Escherichia coli. Transformation is carried out and the obtained transformant is cultured to infect a phage. Thereby, a cDNA bank is constructed and cloned with a probe to select a positive clone. A plasmid is extracted from the obtained clone and treated with a restriction enzyme to afford the objective DNA, containing an amino acid sequence and capable of coding a protein having human-derived glyoxalase I activity.
KIN NANJIYUN
UMEZAWA YURI
