Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

Juan Miguel Puertas, Jordi Ruiz, Mónica Rodríguez de la Vega, Julia Lorenzo, Glòria Caminal, Glòria González

Research output: Contribution to journalArticleResearchpeer-review

14 Citations (Scopus)

Abstract

A high cell density cultivation protocol was developed for the secretory production of potato carboxypeptidase inhibitor (PCI) in Escherichia coli. The strain BW25113 (pIMAM3) was cultured in fed-batch mode employing minimal media and an exponential feed profile where the specific growth rate was fixed by limitation of the fed carbon source (glycerol). Plasmid loss rates were found to be proportional to the specific growth rate. Distribution of PCI along the cell compartments and the culture media was also dependent on the fixed growth rate. When specific growth rate was kept at μ=0.10h-1, 1.4g PCIL-1 were obtained when adding the product present in periplasmic extracts and supernatant fractions, with a 50% of the total expressed protein recovered from the extracellular medium. This constituted a 1.2-fold increase compared to growth at μ=0.15h-1, and 2.0-fold compared to μ=0.25h-1. Last, a cell permeabilization treatment with Triton X-100 and glycine was employed to direct most of the product to the culture media, achieving over 81% of extracellular PCI. Overall, our results point out that production yields of secretory proteins in fed-batch cultures of E. coli can be improved by means of process variables, with applications to the production of small disulfide-bridged proteins. Overall, our results point out that control of the specific growth rate is a successful strategy to improve the production yields of secretory expression in fed-batch cultures of E. coli, with applications to the production of small disulfide-bridged proteins. © 2010 Elsevier Ltd.
Original languageEnglish
Pages (from-to)1334-1341
JournalProcess Biochemistry
Volume45
DOIs
Publication statusPublished - 1 Aug 2010

Keywords

  • Fed-batch cultivation
  • Potato carboxypeptidase inhibitor
  • Secretory expression in E. coli

Fingerprint

Dive into the research topics of 'Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli'. Together they form a unique fingerprint.

Cite this