Recombinant expression, purification, and refolding to bioactive protein L-isoaspartate-O-methyltransferase of buffalo (Bubalus bubalus)

Abstract

The cryopreserved semen is used in assisted reproductive technologies. There is a continued oxidative stress during cryopreservation, causing oxidative damage to spermatozoa. The isoAsp formation during oxidative stress results in the loss of protein function. The protein L-isoaspartate-O-methyltransferase (PIMT) repairs back the isoAsp residues to L-aspartyl, restoring the lost functionality of the affected proteins. The quality of cryopreserved semen could be improved by using PIMT as a supplement to semen extender. The objective of the study was to express biologically active buffalo recombinant PIMT (rPIMT). The ~650 bp PCR-amplified PIMT was cloned and sequenced. The protein was consistently expressed as inclusion bodies in E. coli T7 Express lysS cells and then bulk-purified under denaturing conditions. The protein sequence showed 96.55% identity with the bovine PIMT protein. The purified rPIMT showed a single protein band corresponding to 31 kDa on SDS-PAGE and Western blot. A synthetic isoaspartate-containing peptide served as the substrate to assess enzymatic activity. The effective methylation of iso-aspartate residues in the synthetic peptide substrate served as evidence of PIMT enzyme activity. Thus, the bioactive rPIMT could potentially be added to buffalo semen extender to repair the oxidatively damaged seminal plasma proteins that arise during cryopreservation.