Evaluation Of Elastin-like Polypeptides Fusion Self-assembly Peptide RADA-16 For Hemostasis

Self-assembly peptide RADA-16(RADARADARADARADA)is a small chemical synthesis peptide.1% RADA-16 solution contact with bleeding wound,rapidly assembled into a nanofibers barrier,it can be used to terminate the traumatic hemorrhage of adult hamster brain,spinal cord,femoral artery and liver in 15 s.United States subsidiary of the Massachusetts Institute of Technology developed RADA-16 for hemostasis products.It dependent synthesis,purification is difficult,synthesis is expensive,only used for brain surgery,application scope and beneficiaries are greatly limited,the crux to solve this problem is to reduce the production cost of RADA-16.In this study,we use the genetic engineering technology to connect the different length of human elastin-like peptides coding gene and RADA-16 coding gene,construction RADA-16 human-like fusion protein(hELPs)prokaryotic expression vector and expression in Escherichia coli,An engineered E.coli Strain with high efficiency and stable expression of the fusion protein hELPs-RADA-16 was screened and its hemostatic properties were analyzed.The following results were obtained:(1)were constructed three prokaryotic expression vectors: pET28a(+)-h ELP36-RADA-16 ?pET28a(+)-h ELP60-RADA-16?pET28a(+)-h ELP96-RADA-16;(2)The three plasmids were successfully introduced into Escherichia coli BL21(DE3)competent cells and over expressed,the expression products were named h ELP36 R,h ELP60 R,h ELP96 R,SDS-PAGE?gray sweep analysis showed that the expression levels of h ELP36 R,h ELP60 R and h ELP96 R were 16.35%,17.80% and 21.57%,respectively,and expressed in BL21(DE3)in soluble form,Western-Blot analysis showed that expressed were target proteins;(3)the purity of h ELP36 R,h ELP60 R and h ELP96 R proteins were 58.84%,73.73%,79.2% by two rounds of inverse transition temperature(ITC),further purification of nickel shows that h ELP36 R purity was greatly improved at 200mol/m L imidazole concentration,h ELP60 R purity was 96.91% at 300mol/m L imidazole concentration,h ELP96 R purity was96.37% at 300mol/m L imidazole concentration,but different imidazole concentration didn't have significantly to improve the purity of fusion proteins h ELP60 R and h ELP96R;(4)The cell proliferation?cell migration and cell adhesion experiment was carried out with sterile protein coated 96 cell plate,The results showed that the fusion protein h ELP36 R,h ELP60 R and h ELP96 R did not significantly promote and inhibit cell proliferation?migration and adhesion in vitro;(5)using h ELP36 R,h ELP60 R,h ELP96 R freeze-dried protein membrane to carry out the experiment of mouse liver hemostasis,the positive control groups was treated with gauze and 1% RADA-16 freeze-dried membrane and the tail collagen,the negative control without any treatment,compared with the negative control group,the three fusion proteins had the ability to stop bleeding(P<0.001),h ELP36R?h ELP60 R and h ELP96R?RADA-16?collagen and negative control mean hemostatic times were 27.21±1.92s?18.65±1.97s?15.85±1.21s?14.44±1.33s?21.23±1.84 s and 37.64±1.34 s.The results showed that 3 kinds of fusion protein were prepared with hemostatic effect,hemostatic ability of h ELP60 R and h ELP96 R slightly better than that of collagen,but only h ELP96 R of the hemostatic ability was nearly to RADA-16.