Prokaryotic Expression,purification,and Crystal Growth Conditions Screening Of Alkaline Protease Inhibitor LupI-MSSS

The alkaline metalloproteases MP is extracted from the strain YS-80-122 and belongs to the family of serralysin.Similarly to the other enzymes of this family reported before,there is an inhibitor gene lupI downstream of the MP gene and LupI can completely inhibit the activity of MP.On the basis of the wild-type inhibitor gene,four amino acids were added to the N-terminus of LupI,named LupI-MSSS.The inhibitor gene lupI-MSSS was expressed in E.Coli,and the conditions of fermentation of the inhibitor were optimized by single factor method and response surface methodology.We purified the protein to meet the purity requirements of subsequent crystallization.Finally,the crystallization conditions of LupI-MSSS were screened.The results of the study are as follows:The expression of inhibitor LupI-MSSS in E.Coli :The recombinant plasmid pET28a-lupI-MSSS was constructed by PCR with plasmid pET28-lupI as template.Then,the recombinant plasmid was transformed into E.coli BL21(DE3)for culture.The positive clones were selected for double digestion,and the fragment was about 363 bp,which was consistent with the expected.The expressed protein was analyzed by SDS-PAGE gel electrophoresis.The results showed that the size of the protein was about 11 KDa,which was the same as that of the predicted protein.The activity assay showed that the expressed LupI-MSSS could completely inhibit the activity of MP.Optimization of fermentation conditions for LupI-MSSS in E.Coli:The expression conditions(initial medium pH,inoculation amount,addition time of IPTG,concentration of IPTG,induction time,induction temperature,shaker speed)of the inhibitor LupI-MSSS were optimized by single factor experiment.Three major factors were screened by Plackett-Burman test: addition time of IPTG,concentration of IPTGand induction time.The three main factors above were optimized by response surface methodology and the final fermentation conditions were as follows: the initial pH was 7,the inoculation amount was 2%,the inducer was added after 2 h,the final concentration of IPTG was 0.5 mmol/L,the induction temperature was 37 ?,the induction time was8.5 h,and the shaking speed was 200 r / min.Purification of inhibitor LupI-MSSS : The purity of the inhibitor LupI-MSSS was 20 times the original after a series of purification steps: ultrafiltration,Superdex200 gel filtration chromatography and Q-Sepharose ion exchange chromatography.SDS-PAGE results showed a single target band and the purity of protein samples was up to 99.7% identified with HPLC.The purity meet to the requirements of crystallization test.Crystal growth conditions screening of LupI-MSSS:The crystal growth conditions of the inhibitor LupI-MSSS were screened using the kit of Hampton Research.And there were 22 of these conditions had crystal observed,and the crystal form was consistent.The better single crystals were analyzed by SDS-PAGE electrophoresis,and the results indicates that the crystal observed under the microscope is indeed the protein of the inhibitor.