Studies of single-chain antibody expression in quiescent Escherichia coli.
Quiescent Escherichia coli cells are generated by overexpressing the Rcd transcript in an hns-205 mutant host. The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures. The expression system is based on two plasmids; one carries the product gene expressed from lambdaP(L) under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from lambdaP(R). Shifting the culture from 30 to 42 degrees C induces Rcd expression and product expression simultaneously. Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant. In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD(600)] of 3.5) was 37 mg x liter(-1), compared to a maximum of 13 mg x liter(-1) in the control culture (final OD(600) of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD(600) of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg x liter(-1). A control culture with a similar feed reached an OD(600) of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg x liter(-1). Quiescent cells at an OD(600) of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg x liter(-1) was achieved in 8 h. The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture. This study demonstrates that it is possible to establish high-density quiescent E. coli cultures that are capable of efficient synthesis, folding, and export of proteins.
Mukherjee
K J
KJ
Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom.
Rowe
D C D
DC
Watkins
N A
NA
Summers
D K
DK
D001709:Biotechnology / Q000379:methods; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D005285:Fermentation; D005455:Fluorescent Antibody Technique; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D003470:Culture Media; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv