Processing Of The N-terminal Initiation Methionine From Recombinant Human Interferon-α2b By Escherichia Coli Methionine Aminopeptidase

Objective To construct the prokaryotic expression system of recombinant Escherichiacoli Methionine Aminopeptidase(reMAP) and use the reMAP to remove the N-terminalinitiator Methionine residue from recombinant human interferon-α2b (rhIFN-α2b).Methods The sequences of E.coli map gene was amplified with PCR using thegenome of E.coli BL21as a template and then cloned into the cloning vectorpGEM-7zf(-), transformed into competent cell of E.coli DH5α. and identified byblue-white screen and DNA sequencing, then reMAP gene was cloned into theIPTG-controlled expression vector pACYCDuet-1, the recombinantpACYCDuet-1-MAP vector was identified by Nco I/BamH I digest, PCR identificationand DNA sequencing,then it was transformed into competent cell of E.coli BL21. Thepositive recombinant clones were expressed under IPTG induction，the expressionproductions were investigated by SDS-PAGE,Western blot analysis and activity assay.Then determine the in vitro processing of rhIFN-α2b by purified MAP and the in vivoprocessing by coexpression of MAP and rhIFN-α2b. Results The result of NcoI/BamH I digestion, PCR identification and DNA sequencing showed that therecombinant pACYCDuet-1-MAP vector was successfully constructed; The analysis ofSDS-PAGE and Western blot indicated that both MAP and rhIFN-α2b were expressedcorrectly; Activity assay indicated that the reMAP had the ability to remove theN-terminal methionine from polypeptides and the activity was greater than30pmol/μg/min; The N-terminal sequencing of rhIFN-α2b indicated that the N-terminalinitiator Met removal rate of in vivo processing was above94%while the one of in vitroprocessing was above96%.Conclusion The reMAP could be used to remove theN-terminal initiator Methionine residue from rhIFN-α2b.