Cloning Of Novel Fusion Expression Tags And Construction Of Recombinant Engineering Vectors Of Pseudomonas

Most heterologous proteins exhibit as insoluble or biologically inactive inclusion bodies when overexpressed in Escherichia coli,which hinders the research of its function.Currently,fusion expression is one of the effective ways to improve soluble protein.Protein tag is a polypeptide or protein,expressing with target protein,which refers to the use of recombinant DNA technology in vitro,convenient for access to expression,detection and purification of the target protein.Protein fusion tags including 6x histidine,MBP,GST,eGFP,Flag and SUMO are widely used in the basic research as well as commercial protein productions.However,each tag has its own limitations.This study explored the feasibility of the source of the lambda Beta protein as a novel fusion tag,expressed with four genes,gfp,hRnBp,slr1975 and PMT0106.The beta gene fragment containing the N-terminal histidine tag and C-terminal the TEV cleavage site were cloned to into expression vector pET30(+)to form a general order vector in order to cloning and expression of the target gene.The solubility of hRnBp and slr1975 was significantly improved when fusion expression with Beta tag,and expression yield of Beta-GFP fusion expression was elevated while keeping the solubility,whereas for PMT0106,the fusion expression showed no obvious improvement.The activity of the fusion protein was increased,detecting by Whole-cell catalyzed Neu5Ac biotransformation.We could obtain expected band type when purified fusion proteins were cut by the TEV protease,thus demonstrating the potential use of Beta protein as a novel fusion tag.The lambda Red recombination system has been used to inactivate chromosomal genes in various bacteria and fungi.Recombinase genes include exo(red a)gene,bet(red?)gene,gam gene,exo coding DNA 5 'end to the 3' end of exonuclease Exo generating in the double-stranded DNA molecule 3 'protruding end molecule;bet encoding single-chain binding protein Beta,binding 3 'overhangs generated by the action Exo DNA molecules,and also to exercise the recombinant enzyme,promoting two single-stranded homologous DNA molecules to form the recombinant molecule by annealing or chain invasion.Gam protein can inhibit the degradation of exogenous DNA by the E.coli RecBCD.This study aims to construct a recombinant engineering systems based on plasmid:Red recombinase induced by arabinose,I-SceI induced by IPTG and sucrose vice selectable marker,to achieve the PAO1 strain of non-redundant bases one step chromosomal gene knockout.At present,we have constructed recombineering vectors with three kinds of replicons.