Construction Of High-level Expression System Of Echistatin In E.coli

Objective:Construct high-level expression system of Echistatin in E.coli Methods:Obtain amino-acid sequence of Echistatin from Genebank Database.Considering the bias of usage of 61 available aminoacid codons in E.coli , design the coding sequence of Echistatin , Synthesize the DNA sequence chemically , get single copy coding gene and repeated two copy coding gene of Echistatin .insert the sequence into expression vector PBV220,And more ,we construct fusion expression clone of Echistatin with PCR , Identify the recombinant vector by DNA sequencing.The recombinant vector were transformed into different Escherichia coli(JMI09, DH5a,BL21 (DE3), BL21 (DE3) plySs) as host cell.identify the expressed protein by SDS-PAGE and Western-blotting.Results:Designed and synthesized the coding gene of Echistatin.and constructe its single copy expression vector , Identify the vector by DNA sequencing.but it could not express Echistatin in E.coli.Designed and synthesized the modified coding DNA of Echistatin,and constructe its two copy expression vector , Identify the vector by DNA sequencing,but it could not express Echistatin in E.coli.Constructed the fusion protein expression vector of Echistatin,.and identify it by DNA sequencing.The vector can express Echistatin fusion protein at a high level,the fusion protein molecular weight is about 16000 Da in SDS-PAGE analysis , and the fusion protein consists more than 30% of total protein, thefusion protein has specific antigen-antibody reaction with rabbit anti-Echistatin polyclonal antibody.The fusion protein include:hPK5.His+ 6tag,and Echistatin.Echistatin can be released by CNBr cleavage.ConclusionConstructed the high-level expression clone of Echistatin in E.coli . the expression of recombinant protein is higher, it make the further study of Echistatin feasible.Expressing low molecular weight protein and peptide in E.coli will encouter many problem.such as mRNA Stability , protein degradation, mRNA translational Initial . Fusion protein expression system can overcome those problem, increased the yield of yield of recombinant protein in E.coli. This remarkable increase in protein yield was thought to be due to protection of the target protein from proteolysis, improved folding, and efficient mRNA translation .Fusion protein also make the detection and purification easy ,is a good strategies for achieving high-level expression of genes in Escherichia coli.