TY - JOUR
T1 - Recombinant Fab expression and secretion in Escherichia coli continuous culture at medium cell densities: Influence of temperature
AU - Rodríguez-Carmona, Escarlata
AU - Cano-Garrido, Olivia
AU - Dragosits, Martin
AU - Maurer, Michael
AU - Mader, Alexander
AU - Kunert, Renate
AU - Mattanovich, Diethard
AU - Villaverde, Antonio
AU - Vázquez, Felícitas
PY - 2012/3/1
Y1 - 2012/3/1
N2 - Production of recombinant antibody fragments (Fabs) in Escherichia coli has gained interest because of the recognised advantages of this expression system and because Fabs do not require glycosylation. However, more comprehensive studies on the factors that influence expression conditions and product yield are still required for full process development. In this work, the effect of growth temperature on the periplasmatic expression of the 3H6 Fab in E. coli was studied in carbon-limited continuous cultures operated at medium cell densities. Three different temperatures were assayed, namely 37, 33 and 30 °C. Results showed that biomass yield was not affected within this temperature range whilst product yield increased as temperature decreased. Periplasmic Fab secretion corresponded to 30% of the produced Fab protein and its efficiency was irrespective of the process temperature. Moreover, considerable product leakage to the culture supernatant was detected in all cases, ranging from about 40% at 37 °C to almost 70% at 30 °C. Besides, plasmid loss was observed along process time indicating a selective pressure against plasmid-bearing cells. This study supports the potential of continuous cultivations of E. coli at medium cell densities under well controlled conditions as a tool for characterising the impact of environmental parameters and cell physiology under protein production conditions. © 2011 Elsevier Ltd. All rights reserved.
AB - Production of recombinant antibody fragments (Fabs) in Escherichia coli has gained interest because of the recognised advantages of this expression system and because Fabs do not require glycosylation. However, more comprehensive studies on the factors that influence expression conditions and product yield are still required for full process development. In this work, the effect of growth temperature on the periplasmatic expression of the 3H6 Fab in E. coli was studied in carbon-limited continuous cultures operated at medium cell densities. Three different temperatures were assayed, namely 37, 33 and 30 °C. Results showed that biomass yield was not affected within this temperature range whilst product yield increased as temperature decreased. Periplasmic Fab secretion corresponded to 30% of the produced Fab protein and its efficiency was irrespective of the process temperature. Moreover, considerable product leakage to the culture supernatant was detected in all cases, ranging from about 40% at 37 °C to almost 70% at 30 °C. Besides, plasmid loss was observed along process time indicating a selective pressure against plasmid-bearing cells. This study supports the potential of continuous cultivations of E. coli at medium cell densities under well controlled conditions as a tool for characterising the impact of environmental parameters and cell physiology under protein production conditions. © 2011 Elsevier Ltd. All rights reserved.
KW - Continuous culture
KW - E. coli
KW - Fab production
KW - Process design
KW - Process monitoring
KW - Temperature influence
U2 - 10.1016/j.procbio.2011.11.024
DO - 10.1016/j.procbio.2011.11.024
M3 - Article
SN - 1359-5113
VL - 47
SP - 446
EP - 452
JO - Process Biochemistry
JF - Process Biochemistry
IS - 3
ER -