Application of a Seamless and Restriction Endonuclease-free Cloning Method to Produce Recombinant Full-length N-terminal His-tagged Streptolysin O in E.coli

Background and Aims: DNA cloning, sub-cloning and site directed mutagenesis are the most common strategies in nearly all projects of recombinant protein production. The classical method of restriction site cloning is unsatisfactory due to the need for supply of restriction enzymes and the inefficiency of the digestion reaction. Many new methods, including recombinatorial cloning and ligation independent cloning need additional enzymes and kits. In this project we insert a full-length streptolysin O gene into an expression plasmid without using any uncommon commercial enzymes. Materials and Methods: Steptolysin O gene was amplified by polymerase chain reaction (PCR) and introduced into the pPSG-IBA۳۵ vector using a quick-change PCR. At the same time the gene was double digested and sub-cloned into pET۲۸a (+). Both constructs were introduced into BL۲۱ DE۳ cell. Proteins were purified by Ni-NTA column and hemolytic activity was evaluated by spectrophotometry using human red blood cells. Results: Steptolysin O was subcloned into the pET۲۸a (+) and pPSG-IBA۳۵ vectors and expressed in E. coli. Protein was purified with over ۹۰% purity. The IC۵۰ of C and N terminus his-tagged protein were ۰.۲۲ and ۰.۲۹ µg/ml, respectively in hemolysis assay. Conclusions: This study showed for the first time that full-length streptolysin O can be expressed in E. coli cytoplasm without any toxicity for the bacteria itself. The only additional amino acids expressed on the protein were his-tag. To study the role of this toxin it would be better to express the protein with the same strategy to have minimal extra amino acids on the protein.

Keywords:

Pore forming toxin , Seamless cloning , Streptolysin O , استرپتولایزین O , توکسین منفذساز , کلون سازی seamless