Studies of Single-Chain Antibody Expression in Quiescent Escherichia coli

Abstract

ABSTRACT 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 λP L under the control of the c I857 temperature-sensitive repressor, while the second expresses Rcd from λP 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 [OD 600 ] of 3.5) was 37 mg liter −1 , compared to a maximum of 13 mg 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 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 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 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.