Extracellular production of an anti-HER2 single-chain variable antibody fragment in Escherichia coli

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 λPL under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from λPR. 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.

Download Full-text

Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli

Applied Microbiology and Biotechnology ◽

10.1007/s002530050299 ◽

1994 ◽

Vol 42 (4) ◽

pp. 595-603 ◽

Cited By ~ 4

Author(s):

J. E. Somerville ◽

S. C. Goshorn ◽

H. P. Fell ◽

R. P. Darveau

Keyword(s):

Escherichia Coli ◽

Antibody Fragment ◽

Single Chain ◽

Single Chain Antibody ◽

Single Chain Antibody Fragment

Download Full-text

Expression of VH-linker-VL orientation-dependent single-chain Fv antibody fragment derived from hybridoma 2E6 against aflatoxin B1 in Escherichia coli

Journal of Industrial Microbiology & Biotechnology ◽

10.1007/s10295-014-1570-9 ◽

2014 ◽

Vol 42 (2) ◽

pp. 255-262 ◽

Cited By ~ 19

Author(s):

Aiping Liu ◽

Yang Ye ◽

Weifeng Chen ◽

Xiaohong Wang ◽

Fusheng Chen

Keyword(s):

Escherichia Coli ◽

Aflatoxin B1 ◽

Antibody Fragment ◽

Single Chain ◽

Single Chain Fv ◽

Fv Antibody ◽

Single Chain Fv Antibody

Download Full-text

Broad-Host-Range Plasmid pJB658 Can Be Used for Industrial-Level Production of a Secreted Host-Toxic Single-Chain Antibody Fragment in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.70.12.7033-7039.2004 ◽

2004 ◽

Vol 70 (12) ◽

pp. 7033-7039 ◽

Cited By ~ 55

Author(s):

H. Sletta ◽

A. Nedal ◽

T. E. V. Aune ◽

H. Hellebust ◽

S. Hakvåg ◽

...

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Antibody Fragment ◽

Fine Tuning ◽

Broad Host Range ◽

Heterologous Proteins ◽

Single Chain ◽

Single Chain Antibody ◽

High Cell Density Cultures ◽

Industrial Level

ABSTRACT In industrial scale recombinant protein production it is often of interest to be able to translocate the product to reduce downstream costs, and heterologous proteins may require the oxidative environment outside of the cytoplasm for correct folding. High-level expression combined with translocation to the periplasm is often toxic to the host, and expression systems that can be used to fine-tune the production levels are therefore important. We previously constructed vector pJB658, which harbors the broad-host-range RK2 minireplicon and the inducible Pm/xylS promoter system, and we here explore the potential of this unique system to manipulate the expression and translocation of a host-toxic single-chain antibody variable fragment with affinity for hapten 2-phenyloxazol-5-one (phOx) (scFv-phOx). Fine-tuning of scFv-phOx levels was achieved by varying the concentrations of inducers and the vector copy number and also different signal sequences. Our data show that periplasmic accumulation of scFv-phOx leads to cell lysis, and we demonstrate the importance of controlled and high expression rates to achieve high product yields. By optimizing such parameters we show that soluble scFv-phOx could be produced to a high volumetric yield (1.2 g/liter) in high-cell-density cultures of Escherichia coli.

Download Full-text