Efficient Molecular Biological Manipulations with Improved Strategies Based on Novel Escherichia coli Vectors.

Abstract

In this study, some novel plasmids have been constructed for flexible and zero-background molecular cloning, more efficient expression, and purification of proteins with improved strategies. The plasmids pANY4-pL18-ccdB and pANY4-pR18/pL18-ccdB have different promoters in the complementary DNA strands. Therefore, recombinant plasmids for either isopropyl-β-d-thiogalactoside-induced or temperature-induced protein expression could be simultaneously constructed in a single molecular cloning process for parallel comparison. Intriguingly, the mutated pL18 and pR18/pL18 promoters performed similar to or even better than the T7 promoter when used for promoting the expression of the GFP or pfLamA enzyme. Moreover, the plasmid pANY8 containing the His-elastin-like polypeptide (ELP)-intein multifunctional tag was constructed, and special purification protocol was designed to obtain purified proteins without the requirement of time-consuming dialysis steps to remove imidazole and high concentration of salt ions. Additionally, the urea-based denaturation and refolding processes can be conveniently integrated into the ELP-mediated precipitation protocol for purification of insoluble inclusion bodies, omitting the time-consuming dialysis steps.