Cloning, Expression, Purification And Characterization Of Soluble Recombinant Endoproteainase AspN

Endoproteinase AspN (flavastacin) is a zinc metalloendopeptidase which selectivelycleaves peptide bonds N-terminal to aspartic acid residues. It is ideal for proteomeanalysis by mass spectrophotometry. It is apply for protein and peptide identification.The common endoproteinase AspN are extract from the bacteria excretion,Elizabethkingia meningoseptica and Pseudomonas fragi. Because of low products anddifficult preparation and high cost，they are the limitions of the use of the enzyme.Expression the recombinant endoproteinase AspN is the key of solve the problems.The recombinant endoproteinase AspN have two difficulties: it is difficult to refoldthe protein through expressed as inclusion body; it is poisionous for the host byexpressed as soluble from. Our research expressed two kinds of recombinantendoproteinase AspN,flavastacin and peptidyl-Asp metalloendopeptidase. The pET32aplasmid was used as a template for creating vectors that encode flavastacin undercontrol of the strong bacteriophage T7promoter. The carboxy-terminal poly-histidine tagenable purification by Ni affinity chromatography. The protein was cut its tag byactivating itself. The plasmid peptidyl-Asp metalloendopeptidase/pGEX that encodepeptidyl-Asp metalloendopeptidase under control of strong bacteriophage T7promoter.The carboxy-termina GST tag enabled purification by GST affinity. We got the highpurity by a serious of chromatography. We tested the activity of the protease byfluorescence assay, HPLC and SDS-PAGE. The results demonstrate that both theprotease had the high activity.First reported recombinant endoproteinase AspN, and the recombinant endoproteinaseAspN was produced in E.coli using prokaryotic expression system. The enzyme withhigh expression and it is easy to get by purification. By the fluorescence assay test theenzymes has the character of simple preparation and high product. The recombinantendoproteinase AspN will widely used in MS to improve the coverage of the proteinsequence and put a foundation for the protein function and mechanization research.