中国生物工程杂志

In order to confirm the role that the 49th amino acid residue plays in enzymatic inactivity of Glutamine 49 phospholipase A2 (Gln49-PLA2), site-directed mutagenesis of its 49th amino acid gene codon was conducted using PCR. Aspartic acid 49 phospholipase A2 (Asp49-PLA2--Q49D-PLA2), the mutant of Gln49-PLA2 was expressed in E. coli with pET32a+ vector. The fusion protein, expressed as inclusion body, after being denatured, was on-column refolded and purified by immobilized metal affinity chromatography (IMAC), and then cleaved by Factor Xa. The mature Q49D-PLA2 mutant was obtained by Hitrap SP cation exchange and Superdex 75 gel filtration chromatography, with the recovery rate of 1.3%, and the specific activity of the mature Q49D-PLA2 mutant was 72 U/mg. It has been demonstrated that the 49th glutamine amino acid residue is the main reason in enzymatic inactivity of Gln49-PLA2 and the results are helpful for denatured protein refolding, especially in rich disulfide bonds conditions.