Application of a Seamless and Restriction Endonuclease-free Cloning Method to Produce Recombinant Full-length N-terminal His-tagged Streptolysin O in E.coli
Abstract:
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-IBA35 vector using a quick-change PCR. At the same time the gene was double digested and sub-cloned into pET28a (). Both constructs were introduced into BL21 DE3 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 pET28a () and pPSG-IBA35 vectors and expressed in E. coli. Protein was purified with over 90% purity. The IC50 of C and N terminus his-tagged protein were 0.22 and 0.29 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.
Language:
English
Published:
International Journal of Medical Laboratory, Volume:4 Issue: 3, Aug 2017
Pages:
189 to 200
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