Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Expression and simple purification strategy for the generation of antimicrobial active enterocin P from Enterococcus faecium expressed in Escherichia coli ER2566
Show others and affiliations
2014 (English)In: Iranian Journal of Biotechnology, ISSN 1728-3043, Vol. 12, no 4, 16-25 p., e1154Article in journal (Refereed) Published
Abstract [en]

Background: Enterocin-P of Enterococcus faecium P13 (EntP) is of great interest as a food preservative and medicine due to its non-toxicity and broad antimicrobial spectrum at various pH, as well as its excellent thermal stability. However, recombinant production of EntP is still in laboratory because of low productivity and complex purification process. Objectives: In this study, we aimed to develop efficient methods for high-level expression and convenient purification of the recombinant EntP.Materials and Methods: An artificially synthesized gene (entP) of 132 bp encoding mature enterocin P of E. faecium P13 was cloned in plasmid pTWIN1 under the control of an inducible T7lac promoter for expression of fusion protein EntP- Mxe GyrA mini-intein-chitin binding domain (CBD) (abbreviated by EntP-Int-CBD) in E. coli. Recombinant EntP was released from the fusion protein by DTT digestion and cleaned by distilled water and checked for anti-bacterial activity. Results: The fusion protein was highly expressed in insoluble form in E. coli at 37ÂșC with 0.05 mM IPTG induction. The insoluble fusion protein EntP-Int-CBD was easily prepared by cell sonication and centrifugation to remove soluble con- taminants. The repeat washing steps with Triton X-100 were applied to reduce contaminants. After DTT-induced self- digestion in urea 4 M, the EntP released from the fusion protein was insoluble in water and easier to be separated from sol- uble Int-CBD by centrifugation. The recombinant peptide was soluble in 20% 2-propanol in 0.1% trifluoroacetic acid (TFA) and exhibited strong anti- Listeria monocytogenes and Staphylococcus aureus activities.Conclusions: This study is the first report providing a simple, quick and straight forward procedure for heterologous production of functional and pure Enterocin P without using any chromatography columns in the purification process.

Place, publisher, year, edition, pages
2014. Vol. 12, no 4, 16-25 p., e1154
Keyword [en]
Antimicrobial activity, E. coli expression, Enterocin P, Mxe GyrA mini-intein-chitin binding domain (CBD), Purification
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-166952ISI: 000354656700006Scopus ID: 2-s2.0-84924855830OAI: oai:DiVA.org:kth-166952DiVA: diva2:815063
Note

QC 20150529

Available from: 2015-05-29 Created: 2015-05-21 Last updated: 2017-05-19Bibliographically approved

Open Access in DiVA

No full text

Scopus

Search in DiVA

By author/editor
Enfors, Sven-Olof
By organisation
School of Biotechnology (BIO)
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 59 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf