Prokaryotic Expression And Purification Of Insecticidal Proteins Bt And CpTI

Bacillus thuringiensis(Bt)is the most extensively researched and widely used microbial pesticide across the globe.During the spore formation stage,Bt bacteria can produce Bt crystal proteins that are toxic to Lepidoptera and Diptera insect pests.Currently,the Bt gene has become the most extensively studied and applied gene in plant genetic engineering.Cowpea Trypsin Inhibitor(Cp TI)gene,which has a broad spectrum of insecticidal activity and is less prone to developing resistance,is widely used in plant genetic engineering due to these advantages.To evaluate the safety of insect-resistant gene expression products in transgenic crops,it is essential to obtain a substantial amount of exogenous protein in an in vitro microbial system.The objective of this study is to construct a prokaryotic expression system that can efficiently express and purify the insecticidal genes,Cry1Ab/Ac and Cp TI.The study is divided into two parts:In this subject paper,the initial step involves the expression and purification of the Bt protein in prokaryotes.To achieve this,the exogenous Cry1Ab/Ac gene was transferred into the p ET-28a(+)vector,and the resulting expression vector,p ET28a-Cry1Ab/Ac,was transformed into Escherichia coli BL21(DE3)strain.The optimal cultivation conditions were determined through IPTG induction,and the resulting expression product was confirmed to be Cry1Ab/Ac protein inclusion bodies via SDS-PAGE analysis.The inclusion bodies were purified using a "purification before renaturation" approach,which involved several crucial steps such as inclusion body washing,denaturation and solubilization,and Ni-NTA affinity purification.The final product,a Cry1Ab/Ac protein of high purity,was obtained through a combination of Ni-NTA purification and gel filtration,followed by dialysis and renaturation.The findings suggest that the successful expression and purification of Cry1Ab/Ac protein was achieved when transforming the recombinant vector p ET-28a(+)into BL21(DE3)strain.This provides a dependable framework and support for future research on Bt proteins.The second part of this study involves the expression and purification of the Cp TI protein in prokaryotes.To achieve this,we designed primers that would eliminate the first 27 AA amino acids of the Cp TI protein sequence.Additionally,we added a 15-25 bp fragment that was homologous to the vector on the 5’ end as a homologous arm.We also optimized the codons based on the degeneracy of the codons.The linearized vector was constructed by cutting the p Smart-I plasmid vector with Bamh I and Xho I restriction enzymes,and the target sequence was seamlessly cloned into the Xho I site of the p Smart-I expression vector based on homologous recombination.Then,the recombinant plasmids were transformed into TOP10 E.coli,and after verifying the recombinant plasmids by double enzyme digestion,the correct recombinant plasmids were transformed into BL21(DE3)expression strain for induction expression by 0.5 m M IPTG at 37℃.The results of SDS-PAGE analysis indicated that the target protein was successfully expressed under the induction conditions.To further optimize the expression conditions,this study compared the expression temperature at 37℃ and 15℃ and the effect of IPTG concentration of0.2m M and 1.0m M on the expression of the target protein.The results showed that the target protein could be solved in the solution under different induction conditions.Finally,the purified target protein was obtained through Ni-NTA affinity purification and SUMO cleavage and refiltration to remove the tag,and the final purity was as high as 96%.In summary,this study effectively utilized modern biotechnological methods to express and purify two crucial insecticidal proteins,namely Cry1Ab/Ac and Cp TI.The proteins underwent thorough analysis and detection,laying a solid groundwork for future research and practical applications.Additionally,this study serves as a dependable reference for similar research,and holds immense practical value.