Cloning and expression of rec-pediocin CP2 in Escherichia coli using OmpA and TAP gene fusion approach Kumar Balvir*, Balgir P. P., Kaur B. Department of Biotechnology, Punjabi University, Patiala, Punjab, India *Email: balvirkumarbt@gmail.com, balvirkumar@yahoo.co.in
Online published on 19 March, 2013. Abstract Study was aimed at cloning, expression and characterization of rec-pediocin CP2 in Escherichia coli BL21(DE3) using ompA and tap gene fusion expression approach. pET32(b)-pedA vector containing an inframe fusion of sequences encoding E. coli ompA secretion signal, two tandem affinity purification tags and rec-pediocin synthetic gene was introduced into a pediocin CP2 resistant strain of E. coli BL21(DE3). Rec-protein was accumulated in periplasmic fraction as well as inclusion bodies of transformed E. coli cultures and was extracted by cell lysis. Crude fusion protein did not show any biological activity. Purification of rec-pediocin involved two tandem affinity chromatographic steps based on 6XHis-tag and Strep-tag. In between these, enterokinase cleavage was carried out. After digestion, rec-pediocin displayed a very strong bactericidal activity against Listeria monocytogenes. The process based on T7-driven pET expression system and tandem affinity chromatography resulted in approximately 10 times higher yield of rec-pediocin (in terms of specific activity) as compared to the native P. acidilactici MTCC5101. Top Keywords Rec-pediocin, Pediococcus acidilactici, Pediocin CP2, Heterologous expression, Cloning, Bacteriocin. Top |