High Expression Of Recombinant Human Platelet Factor 4 In Escherichia Coli And Its Purification,Characterization

Purpose1 Construction of prokaryotic high expression vector of human Platelet Factor 4(h PF4)2 Expression and purification of r h PF43 Bioassay of r h PF4 MethodsAccording to the modulation character of eukaryotic protein expression in prokaryotic cells ,We design a pair of particular primers, and construct a prokaryotic expression vector PBV220- r hPF4 by DNA polymerase chain reaction(PCR) and DNA recombinant technic .The expression plasmid was identified with PCR and DNA sequencing.PBV220- r hPF4 was transformed into E.coli DH5a , BL21(DE3) and induced by increasing the temperature to 42 ℃. We identified the expression protein by SDS-PAGE and Western-blotting. After the expression form analysis,The soluble recombinant protein part was purified with heparin-agarose affinity column and a C18 column on reverse phase high performance liquid chromatography(RP-HPLC). Insoluble recombinant protein part was purified by destraction and abstersion of inclusion body. To study the renatural condition of r hPF4 ,We adopted air oxygenation method to make r hPF4 refolding correctly. Inhibition experiment of blood vessel proliferation in the Chicken chorioallantoic membrane was used to study the biocharacter of r hPF4 and renaturation effect.Results1 DNA sequencing demonstrated that we have constructed the expressionplasmid PBV220- r hPF4 successfully. 3'-UTR of h PF4 c DNA was deleted and TAG was mutated to TAATAA .2 After SDS-PAGE and Densitometric scan analysis, the result show that expression level is 25-30% of total bacterial proteins. Western-blotting result demonstrated rhPF4 had specific reaction with rabbit anti- hPF4 antibody.3 r hPF4 was expressed as inclusion body in E.coli,After washing with reagent I(50Mm Tris-Cl Ph 8.0; 100mMNaCl; 0.5Mm EDTA; 2M carbamide; 0.2% TritonX-100; 0.2% DOC) and reagent II (50mol/L, pH8.0 Tris. Cl; 100mM NaCl ;0.5mmol/L EDTA ; 4mol/L carbamide; 0.2% (v/v) TritonX-100; 0.2%DOC) ,We got r hPF4 with the purity of 85%.The yeild is 120mg recombinant protein per litre culture .4 The primary result of Inhibition experiment of blood vessel proliferation in the Chicken chorioallantoic membrane demonstrate that r hPF4 prepared by our methods has bioactivity like wide hPF4.ConclusionWe have constructed the expression plasmid PBV220-hPF4 successfully. 3'-UTR of h PF4 c DNA was deleted and TAG was mutated to TAATAA by PCR. After SDS-PAGE and Densitometric scan analysis, the expression level of r hPF4 is 25-30%. Western-blotting result demostrated rhPF4 had specific reaction with rabbit anti- hPF4 antibody.Our system improve the expression level of r hPF4 by 80 fold compared with PT7-7- r hPF4. After purified and renatured, r hPF4 prepared by our methods has bioactivity like wide hPF4. Our study establish a stable base for further reseach of the h PF4 and provide a theoretics gist for modulative mechanism of eukaryotic protein expression in prokaryotic cells.