Prokaryotic Expression & Denaturation And Renaturation Of Three Single-Residue Variants Of Bovine Prion Protein

Objective: It is to construct recombinant plasmids about one normal recombinant and three single-residue variants of bovine prion protein(PrP) from their gene clones, which are pGEX-BoPrP(93-241),pGEX-BoPrP(93-241)(S154N),pGEX-BoPrP(93-241)(A129V) and pGEX-BoPrP(93-241)(Q234R), and their recombinant E.coli GST-BoPrPs, thus 4 active recombinant bovine PrPs can be obtained.They will be used in the research of the relation of PrP gene mutations to the structures and properities of PrPs. Method: Adopting the recombinant DNA technology, the prokaryotic expression plasmids of one normal recombinant and tree single-residue variants of bovine prion protein were constructed. Then they were transformed into E.coli BL21(DE3), and the recombinant E.coli GST-BoPrP(93-241), GST-BoPrP(93-241)(S154N), GST-BoPrP(93-241)(A129V) and GST-BoPrP(93-241)(Q234R) were obtained, respectively. After optimizing the expression conditions and inducing the recombinant bacteria, four recombinant GST-BoPrPs were expressed. When the bacteria were dissolved, the inclusion bodies were extracted with 0.1% Triton X-100 and 4 mol/L urea. The extracted products were denatured with 8 mol/L urea, and the denatured products were refolding to form correct structures by dialysis in 4℃. At the same time, renatured GST-BoPrP(93-241)(Q234R) was purified through GSTrap FF column, and the effects of purification and renaturation were identified by SDS-PAGE and Western blotting. Results: The recombinant E.coli GST-BoPrP(93-241), GST-BoPrP(93-241)(S154N), GST-BoPrP(93-241)(A129V) and GST-BoPrP(93-241)(Q234R) had been constructed successfully, and they could produce the expectant molecular size about 42.4 ku of fusion proteins responsed with the anti-bovine PrP monoclonal antibody 4C11, respectively. Under the optimized expression condition(37℃, 1 mmol/L IPTG, induced for 6 h), 4 recombinant expressed products were account for 30%～45% of overall bacterium proteins severally. The expressed expectant proteins are inclusion bodies in bacteria. The identified result confirmed the renatured products had higher purity than the denatured products, when the proteins in inclusion bodies had been purified, denatured with urea after being released from bacteria, and renatured by dialysis. The renatured products are the expectant molecular size about 42.4 ku of proteins responsed with the anti-bovine PrP monoclonal antibody 4C11, respectively. After being purified through GSTrap FF column, the purified GST-BoPrP(93-241)(Q234R) is the expectant molecular size about 42.4 ku of protein, and it has immunoreactivity. The comparison of the renatured products with the purified product revealed: the contaminative proteins about 31~40 ku were removed, but there was a new line about 26 ku in gel. The result showed the expectant product had higher purity through GSTrap FF column than the renatured products.