Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.
The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.
Chalmers
J J
JJ
School of Chemical Engineering, Cornell University, Ithaca, New York 14853.
Kim
E
E
Telford
J N
JN
Wong
E Y
EY
Tacon
W C
WC
Shuler
M L
ML
Wilson
D B
DB
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D002479:Inclusion Bodies / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010957:Plasmids; D013696:Temperature; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
D004269:DNA, Bacterial; D004815:Epidermal Growth Factor; D001618:beta-Lactamases