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High-yield production of recombinant CRM197, a non-toxic mutant of diphtheria toxin, in the periplasm of Escherichia coli. 2017

Philippe Goffin, and Marianne Dewerchin, and Philippe De Rop, and Normand Blais, and Philippe Dehottay
GSK Vaccines, Rixensart, Belgium.

A high cell density fed-batch process was developed for production of recombinant CRM197, a non-toxic mutant of diphtheria toxin widely used as a carrier in polysaccharide-protein conjugate vaccines. Fully soluble recombinant CRM197 was obtained in high yields and with an authentic N-terminus, by targeting the protein to the periplasm of Escherichia coli using the Signal Recognition Particle (SRP)-dependent signal sequence of FlgI. Response Surface Methodology (RSM) was used to optimize the set-points of key process parameters (pH and feed rate at induction). Optimal production of periplasmic CRM197 was found at a slightly basic pH (7.5). The feed rate during induction was positively correlated with the accumulation of unprocessed cytoplasmic CRM197, consistent with limited capacity of the SRP secretion pathway. Decreasing the feed rate to align the protein synthesis rate with the secretion capacity, resulted in minimal production of cytoplasmic CRM197. Besides, the host background was found critical for production of periplasmic CRM197: B834(DE3) was the highest producer (>3 g/L), while BLR(DE3) produced one third less CRM197, and very low yields (290 mg/L) were obtained with HMS174(DE3). The optimized process is robust and linearly scalable, and represents a 20-fold yield improvement compared to a process based on Corynebacterium diphtheriae.

UI MeSH Term Description Entries
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005285 Fermentation Anaerobic degradation of GLUCOSE or other organic nutrients to gain energy in the form of ATP. End products vary depending on organisms, substrates, and enzymatic pathways. Common fermentation products include ETHANOL and LACTIC ACID. Fermentations
D006863 Hydrogen-Ion Concentration The normality of a solution with respect to HYDROGEN ions; H+. It is related to acidity measurements in most cases by pH pH,Concentration, Hydrogen-Ion,Concentrations, Hydrogen-Ion,Hydrogen Ion Concentration,Hydrogen-Ion Concentrations
D001426 Bacterial Proteins Proteins found in any species of bacterium. Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial
D061249 Batch Cell Culture Techniques Methods for cultivation of cells, usually on a large-scale, in a closed system for the purpose of producing cells or cellular products to harvest. Batch Culture Techniques,Batch Culture,Continuous Batch Culture,Fed Batch Culture Techniques,Fed-Batch Culture,Fed-Batch Culture Techniques,Shake-Flask Culture,Batch Culture Technique,Batch Culture, Continuous,Batch Cultures,Batch Cultures, Continuous,Continuous Batch Cultures,Culture Technique, Batch,Culture Technique, Fed-Batch,Culture Techniques, Batch,Culture Techniques, Fed-Batch,Culture, Batch,Culture, Continuous Batch,Culture, Fed-Batch,Culture, Shake-Flask,Cultures, Batch,Cultures, Continuous Batch,Cultures, Fed-Batch,Cultures, Shake-Flask,Fed Batch Culture,Fed-Batch Culture Technique,Fed-Batch Cultures,Shake Flask Culture,Shake-Flask Cultures,Technique, Batch Culture,Technique, Fed-Batch Culture,Techniques, Batch Culture,Techniques, Fed-Batch Culture
D018271 Signal Recognition Particle A cytosolic ribonucleoprotein complex that acts to induce elongation arrest of nascent presecretory and membrane proteins until the ribosome becomes associated with the rough endoplasmic reticulum. It consists of a 7S RNA and at least six polypeptide subunits (relative molecular masses 9, 14, 19, 54, 68, and 72K). SRP (Signal Recognition Particle),Particle, Signal Recognition,Recognition Particle, Signal
D019897 Periplasm The space between the inner and outer membranes of a cell that is shared with the cell wall. Periplasmic Space,Periplasmic Spaces,Periplasms,Space, Periplasmic,Spaces, Periplasmic

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