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Expression and Purification of Fusion-typed Pediocin PA-1 in Escherichia Coli

《南京理工大学学报》(自然科学版)[ISSN:1005-9830/CN:32-1397/N]

Issue:
2011年02期
Page:
268-272
Research Field:
Publishing date:

Info

Title:
Expression and Purification of Fusion-typed Pediocin PA-1 in Escherichia Coli
Author(s):
SONG Jian-min12SUN Ai-you1WEI Dong-zhi1
1.State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China;2.Department of Biological and EnvironmentalEngineering,Hefei University,Hefei 230022,China
Keywords:
pediocin PA-1 escherichia coli fusion protein expression purification
PACS:
Q78
DOI:
-
Abstract:
To effectively express pediocin PA-1(PED)in Escherichia coli,the primers specific for PED coding sequence is designed and synthesized according to the National Center for Biotechnology Information.The gene fragment of PED is amplified from genomic DNA of pediococcus acidilacticii LH31 by polymeras chain reaction.The plasmid pET32c-PED,encoding PED fused with His-tagged thioredoxin protein,is constructed and introduced into escherichia coli BL21(DE3)strain.The fusion protein is expressed in inclusion bodies counting 28% of total cellular proteins in the strain after induction of isopropyl-β-D-thiogalactopyranoside(IPTG)and purified by nickel-nitrilotriacetic acid(Ni-NTA)metal affinity chromatography.For the recovery of biologically active PED,the purified fusion protein is cleaved by enterokinase and the liberated PED is finally purified by ultrafiltration with a 78% yield.The fusion protein,which does not have bactericidal activity against enterococcus faecalis,is cleaved by enterokinase and the cleaved PED recovers its own bactericidal activity.

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Last Update: 2012-04-30