Electronic Journal of Biotechnology
versión On-line ISSN 0717-3458
HALAMI, Prakash M y CHANDRASHEKAR, Arun. Heterologous expression, purification and refolding of an anti-listerial peptide produced by Pediococcus acidilactici K7. Electron. J. Biotechnol. [online]. 2007, vol.10, n.4, pp. 563-569. ISSN 0717-3458.
The fusion protein, 6XHis-Xpress-PedA was constructed and expressed in Escherichia coli BL21 (DE3). The presence of a 12.8 kDa recombinant protein, localized in inclusion bodies (IBs) at high concentration, was confirmed by SDS-PAGE analysis and by western blotting using anti-His antibody. The rec-pediocin was purified by Nickel-nitrilotriacetic acid beads and refolded using 5 mM of β-mercaptoethanol along with 1 M glycine. Results indicated that the refolded rec-pediocin had an early elution profile in the RP-HPLC when compared to the unfolded protein and it exhibited biological activity against Listeria monocytogenes V7 which was approximately 25 times less active compared to native counterpart. The final yield of purified rec-pediocin was 3 mg/l of the culture and is estimated to be 8-10 times higher than the purification by conventional methods.
Palabras llave : fusion protein; inclusion bodies; in vitro refolding; pediocin PA-1; Pediococcus acidilactici; RP-HPLC.