Scale-up and optimization of the low-temperature inducible cspA promoter system.
The performance of the major Escherichia coli cold-shock promoter in directing the synthesis of recombinant proteins at low temperatures was investigated in batch fermentations using a plasmid-encoded transcriptional gene fusion between the cspA promoter region and the lacZ gene. Rapid synthesis of beta-galactosidase was observed when the fermentation broth was chilled to 15 degreesC using a variety of cooling profiles, including one modeling the heat-transfer characteristics of a 60-L pilot plant unit. A linear cooling rate of 0.5 degreesC/min led to optimum recovery yields. For all single-temperature downshift experiments, however, the promoter became repressed 60-120 min after initiation of cooling. Both temperature cycling between 15 and 25 degreesC and stepwise temperature downshifts between 37, 29, 21, and 13 degreesC led to multiple inductions of the cspA promoter. Nevertheless, high-efficiency reinduction was only observed during the first temperature pulse when the former strategy was used and when the cells were held at intermediate temperatures for less than 60 min or more than 120 min in the case of successive downshifts. Promoter repression was abolished in host cells bearing a null mutation in the gene encoding the ribosomal binding factor RbfA, leading to the constitutive and high-level expression of beta-galactosidase for 7 h postshift when shake flask cultures were transferred from 42 to 23 degreesC. The suitability of rbfA cells for cspA-driven recombinant protein production was confirmed in high-density fed-batch fermentations. Our results are consistent with the existence of a cold-shock-induced repressor molecule that must accumulate at a threshold concentration before interfering with the production of proteins placed under cspA transcriptional control.
Department of Chemical Engineering, University of Washington, Box 351750, Seattle, Washington 98195, and Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, Washington 98121, USA.
J A
JA
Vasina
M S
MS
Peterson
F
F
Baneyx
D001426:Bacterial Proteins / Q000235:genetics*; D001709:Biotechnology; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005285:Fermentation; D015870:Gene Expression; D005798:Genes, Bacterial; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D007763:Lac Operon; D009154:Mutation; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism; D012269:Ribosomal Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; C092748:RbfA protein, E coli; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012269:Ribosomal Proteins; C062260:cold shock protein CS7.4, Bacteria; D005966:Glucuronidase