Expression, Purification And Structural Prediction Of Cattle CXCR4 And ProTα Gene

Chemokine receptor 4(CXCR4),namely by LESTR,Fusin and CD184,is an important member of chemokine receptor superfamily.It is confirmed that CXCR4 plays key roles in immunological regulation,tumor migration and development,embryological development, programmed cell death,and so on.Furthermore,CXCR4 is a co-receptor for HFV-1 virus entry into CD4⁺T lymphocyte.Recently,CXCR4 has been one of the hot spots of scientific research and therapy for tumor-associated diseases and HIV.Prothymosinα(ProTα) is a highly acidic protein,which is widely distributed and highly conserved among mammals.ProTαmay have similar even much stronger biological activities.On the one hand,ProTαcan promote lymphocytes increase,on the other hand possesses immunological regulation activities.ProTαhas wide prospect in immunological regulation,tumor, self-immune diseases,virus infection,chronic hepatitis,and so on.Moreover,it is a kind of ideal immunological adjuvant for DNA vaccine.In order to prepare CXCR4 antibody and reveal the mechanism of combination between CXCR4 and SDF-1,with the recombinant clone vector pMD18-T-CXCR4 constructed before,the CXCR4 full sequence and its 1-126 bp fragment at 5' terminal(CXCR4^42aa) were successfully inserted to the expression vector pGEX-4T-1,pET-28a(+),respectively,namely by pGEX-4T-1-CXCR4,pGEX-4T-1-CXCR4^42aa and pET-28a(+)- CXCR4.Recombinant E.coli (BL21) was induced with IPTG to express recombinant protein.SDS-PAGE analysis showed that the optimal temperature,concentration of IPTG,induction time for the recombinant pGEX-4T-1-CXCR4^42aa were 37℃,0.8 mmol/L,6 hours,respectively,and the recombinant protein existed mainly in the form of inclusion bodies.The fusion protein,approximately 31 ku in MW,almost made up 45%of the total proteins.After purification,the recombinant protein was observed to react with mouse anti-GST monoclonal antibody by Western blot.However,the recombinant proteins were not expressed in pGEX-4T-1-CXCR4 and pET-28a(+)-CXCR4 even under differential conditions.The probable reasons included:(1) many rarely-used codons in cattle CXCR4 gene;(2) the incompatibility between plasmid and gene;(3) the cytotoxicity of the expressed protein.Furthermore,transmembrane regions,conserved motifs and tertiary structure of the recombinant protein were predicted using bioinformatics and molecular biological softwares. These work would lay the foudation for the study of structural and functional research of CXCR4 molecular and immunotherapy.Based on the recombinant clone vector pMD18-T-ProTαconstructed before,the recombinant expression vectors pGEX-4T-1-ProTαand pET-30α(+)-ProTαwere constructed. After induction by IPTG under differential situations,the recombinant proteins with the molecular weight of 24 ku and 30 ku,respectively,were expressed by the recombinant BL21 codon plus-pET-30a(+)-ProTα.SDS-PAGE showed that the optimal temperature,concentration of IPTG,induction time for this recombinant bacteria were 37℃,0.5 mmol/L,3 hours,respectively, and the recombinant proteins existed mainly in the form of supernatant.The fusion proteins almost contained 30%of the total proteins.After purification,the special immunological reaction was observed between the expression proteins and the rabbit polyclonal antibody against amino acids 1-50 at the N-terminus of PTαprecursor of human origin by Western blot.The expressed proteins were much bigger than the predicted MW value,and two stripes were observed.Maybe these phenomenon were related to the molecular characteristic of cattle ProTα.The definite reasons would be further studied and discussed.So as to obtain lots of recombinant active protein,recombinant P.pastoris GS115-pPIC9K-ProTαwas constructed.The high-copy recombinants were screening by G418 concentration gradient method and PCR method.Then the recombinants were induced by methanol to express the recombinant protein.SDS-PAGE showed that the recombinant protein with the MW of 20 ku was expressed in the supernatant.,whose yield peaked at 120 h after induction.The expressed protein was much bigger than the predicted MW value.This may be related to protein post-translational modification for P.pastoris and the molecular characteristic of cattle ProTα.The recombinant protein was purified by Sephadex G-100.Sequence and structural prediction of the recombinant protein was carried out by using bioinformatics software.This would facilitate to analyze the bioactivity of cattle ProTαprotein and its production and application.