RECOMBINANT STRAIN, E. COLI JH53 (KCTC 8971P) FOR EXPRESSING THERMOSTABLE CYTIDINE DEAMINASE

摘要

PURPOSE: Provided is a gene coding thermostable cytidine deaminase. And a recombinant strain, E. coli JH53 (KCTC 8971P), expressing the same is also provided. The enzyme eliminates only (+) isomer of (+)(-) isomers of glucose so that (-) isomer useful as an ingredient of medications against type B hepatitis virus can be easily separated. CONSTITUTION: A gene coding thermostable cytidine deaminase and a recombinant strain, E. coli JH53 (KCTC 8971P), expressing the enzyme are obtained by the following steps of: i) synthesizing complimentary base sequences of 5'-ACACGGATCCAATTCTAATTTTTCTGTTACATTTTTG-3' and 5'-ACACTGCAGGATTTTCCTACGTTCGGTCTTCC-3' to use them as primers in cloning cdd gene of Bacillus caldolyticus; ii) culturing Bacillus caldolyticus in Luria-broth at 65 deg.C; iii) isolating chromosome DNA by Maniatis T. method; iii) performing PCR using Tag DNA polymerase; iv) cloning foreign gene then inserting it into pDIA5304 vector to recover plasmid pCJH53; v) transforming E.coli JF611(cdd-) with the plasmid pCJH53 to obtain E.coli JH53(KCTC 8971P); vi) culturing the transformant in AB minimal medium at 37 deg.c; vii) centrifuging the cell and followed by washing with cytidine deaminase buffer solution twice and ultra-sonificating for 45 seconds twice; viii) heat-treating the supernatant at 65 deg.C for 15 minutes and performing anion exchange chromatography and Sephacryl S-2oo liquid chromatography.