Abstract: Wolbachia surface protein (WSP) is an Outer membrane protein of gram negative bacteria Wolbachia. WSP has shown its functional role in immune response, cell proliferation, pathogenicity and controlled cell death program. The WSP gene has previously been cloned, but the expression of the protein is low and the purification is time consuming. Therefore the E. coli WSP gene was cloned into the pET19b vector to improve the protein expression and simplify the purification. The WSP gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Wolbachia genomic DNA. The primers were designed with NdeI or Xho 1 restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of WSP. The gene was cloned into the expression vector pET19b through NdeI and Xho 1 restriction endonuclease sites. The resulting plasmid containing WSP was transformed into the bacterial strain BL21 (DE3).The transformed cells were utilized to grow and express the histidine-tagged and the protein was purified using Ni-NTA affinity chromatography.SDS/PAGE gel analysis has shown that the protein was 69% pure and has approximate subunit molecular weight of 25 kDa. The protein purification is completed in one day and 1 liters of culture produced approximately 18 mgs of protein, an improvement on the previous protein expression and purification.
Keywords: Wolbachia surface protein (WSP), Apoptosis, Protein expression and purification, E.coli strain BL21 (DE3), Ni-NTA affinity chromatography.
Title: Cloning, Expression, and Purification of Histidine-Tagged Wolbachia surface protein
Author: Uday Jayaramaiah, Hariprasad T P N, Deekshith Kumar N, Deepak K J, Puttaraju H P
International Journal of Life Sciences Research
ISSN 2348-313X (Print), ISSN 2348-3148 (online)
Research Publish Journals
