Development and application of expression vector based on Ped plasmid of pediococcus acidilactici MTCC 5101

Abstract

The expression vector pPBP5 (5055bp) was developed from plasmid pCP289 of Pediococcus acidilactici MTCC 5101. This lactic acid bacterial strain is a pediocin (pediocin CP2) producer strain and this pediocin production property served as a food grade selection marker for the construction of expression vector pPBP5. The two fragments ped operon (3494bp) and Ori (1561bp) were isolated from plasmid pCP289 on the basis of known sequence of plasmid pSMB74 of Pediococcus acidilactici H. The ped operon encodes the 3494bp pap (pediocin CP2 production) gene cluster consisting of four genes, papA, papB, papC and papD. The cluster has a common promoter and a common rho-independent stem-loop terminator. Each gene has independent ribosomal binding site (rbs) and initiation and termination codons. The three spacer sequences between the genes ranged from 23 to 98 bp with the largest one between papC and papD. The four genes encode four proteins containing 62, 112, 174 and 724 amino acids respectively. The Ori gene cluster involved in plasmid replication and maintenance frequently found in theta-replicating plasmids from lactic acid bacteria. Multiple cloning sites consist of three restriction sites Nhe1, BmtI and Bpu1102I in the developed vector pPBP5. The fibrin polymerization pocket (FPP) gene was designed according to the coding sequences of Pediococcus acidilactici and addition of of NheI and Bpu1102I restriction sites. Ribosome binding site and poly his tag sequence were added to the N terminal and C terminal of the FPP gene sequence respectively. The designed FPP gene was cloned into developed vector pPBP5 and further expressed FPP protein was purified using Immobilized Metal Affinity Chromatography.