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Applied and Environmental Microbiology, May 2004, p. 3005-3012, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.3005-3012.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Studies of Single-Chain Antibody Expression in Quiescent Escherichia coli

K. J. Mukherjee,^1, D. C. D. Rowe,¹ N. A. Watkins,² and D. K. Summers^1*

Department of Genetics, University of Cambridge, Cambridge CB2 3EH,¹ Division of Transfusion Medicine, Department of Haematology, University of Cambridge, Cambridge CB2 2PT, United Kingdom²

Received 27 October 2003/ Accepted 31 January 2004

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 P_L under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from P_R. Shifting the culture from 30 to 42°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 [OD₆₀₀] of 3.5) was 37 mg liter^–1, compared to a maximum of 13 mg liter^–1 in the control culture (final OD₆₀₀ of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD₆₀₀ of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg liter^–1. A control culture with a similar feed reached an OD₆₀₀ of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg liter^–1. Quiescent cells at an OD₆₀₀ of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg 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.

Present address: Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.