Expression And Purification Of PH Responsive Elastin-like Polypeptide

Background:Proteins play important roles in cell signal transduction,molecular transport,metabolism and adhesion.We need to get amount of highly purified protein to explor its structure and function.With the development of genetic engineering technology,the synthesis and expression of recombinant proteins almost become easily.Chromatography is the most common method used in protein separation and purification.Although chromatography has the advantages of high resolution and specificity,it also has many disadvantages such as low production efficiency,high cost,and not easy to be applied in industrial production.The inverse transition cycling(ITC)technique of elastin-like polypeptide(ELP)does not require the chromatographic columns.It can reduce the cost of purification,simplifie the purification process and improve the purity and yield of protein.However,some temperature-sensitive proteins will lose their biological activity at high temperature during purification process.If we want to reduce the inverse temperature of thermo-responsive ELP,we have to find suitable guest amino acids and increase the concentration of ELP monomer and salt ion.It will greatly increases the difficulties of design and purification of ELP.Objective: In order to reduce the difficulty and cost of recombinant protein purification and expand the application of purified recombinant protein with ELP tag,p H-responsive ELPs with a repeat unit sequence of(VAPGHG)3(VAPGSG)were designed in this study,include ELP[H3S]20,ELP[H3S]30,ELP[H3S]40,ELP[H3S]50,ELP[H3S]60.The optimal condition of expression and purification of p H-responsive ELPs were investigated.Methods: The prokaryotic expression plasmids of p ET-ELP[H3S]n(n=10?20?30?40?50?60)were constructed by using OEPCR and RDL technologies and auto-induced by using E.coli.Target proteins were purified with 0.1 M HAc / Na Ac(p H 4.0)and 1.5 M Tris-HCl(p H 8.8)5 M Na Cl.ELP20 as a purified tag was used to construct to tri-block fusion proteins,such as ELP20-12X-20 and ELP20-24X-20.Results: The results showed that ELP10 was not responsive to p H.The purification of ELP20~60 were all over 95 %.The protein yield of ELP20 was higher than others.It could be 75 % afer two rounds of ITC.Without dispersion bands to prove that ELP20 was not degraded during the purification process.The protein yield of ELP30 was 70 %.The appearance of dispersion bands showed that ELP30 was degraded during the purification process.The protein yield of ELP40,ELP50 and ELP60 were 62 %,60 % and 54 %,respectively.With the increased number of unit repeats,the degree of protein degradation raised and protein yield decreased gradually.With two rounds of ITC,the purification of ELP20-12X-20 and ELP20-24X-20 were over 95 %,and the protein yields were 60 % and 66 %,respectively.Conclusions: 1.The prokaryotic expression plasmids of p ET-ELP[H3S]n(n=10?20?30?40?50?60)were constructed by using OEPCR and RDL technologies.The expression of the target proteins could be more than 40 % by optimizing the expression conditions.2.It proved that the sensitivity of p H-responsive ELP to environmental p H and salt ion were affected by the number of repeats.The proteins with more repeats were more sensitive to p H and salt ion,however,the protein yields was reduced.3.The results showed that p H-responsive ELP could be used as a tag for purification of thermo-responsive ELP.It is expected that this method can be used to purify more fusion proteins.