PLAN FOR EXPRESSION AND PURIFICATION ALIPHATIC AMIDASE FROM Rhodococcus erythropolis PR4

Abstract

In order to investigate the expression level in E. coli BL21 DE3 of gene encoding amidase amplified from Rhodococcus erythropolis PR4 DNA genome, we expressed and purified recombinant amidase from E. coli BL21 DE3. 1038 bp amidase – encoding sequence was published on Gene bank with its accession being ACNO01000111. After designing primer and amplifying successfully gene, we cloned in E. coli DH5α and expressed in E.coli BL21 DE3 based on using pGEM-T và pET28a vectors. Having a mass of approximately 38 kDa, amidase was expressed optimally in  E. coli  using 1 mM IPTG as an inducer, at 37^oC, for 4 h. We were successful in purification of recombinant enzyme amidase using Macro-Prep High Q (BIO-RAD) and superdexTM 200 10/300 (GE Healthcare) columns.