Studies of Single-Chain Antibody Expression in Quiescent 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 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 [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.Escherichia coli continues to occupy center stage as a host for large-scale recombinant protein production (21). This is especially true for simple proteins, where posttranslational modifications are not essential for biological activity. Recent developments in our understanding of protein folding have allowed the expression of many proteins in their biologically active soluble form rather than as inclusion bodies (2,3,19). Coupled with more efficient secretion mechanisms, it is now possible to obtain a recombinant protein as a periplasmic or even extracellular fraction, which significantly eases the problems of downstream processing (4,14). While sophisticated vectors with strong and tightly regulated promoters have been developed to maximize product expression, comparatively little attention has been paid to the bacterial host. An improved host could potentially offer gains in product quantity and quality and could be used in conjunction with a wide range of existing vectors.We have been exploring the conflict between the biotechnologist's desire for more and better product and the inclination of the bacterium to produce biomass. In an attempt to shift the balance in favor of the biotechnologist, we developed the quiescent-cell (Q-cell) expression system, in which a plasmid-encoded protein is expressed in nongrowing but metabo...

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

“…Several reports have shown that the PelB signal peptide can successfully secrete scFvs into the periplasm or medium (31,38,39). However, in our study, use of a commercial expression vector with the PelB signal peptide after transformation with PelB-V L -V H or PelB-V H -V L resulted in no growth of either E. coli strain BL21(DE3) or Rosetta (DE3) on selection plates.…”

“…Consequently, two types of C4 scFvs that were 723 bp long with different orientations of the heavy and light chain variable regions (V L -V H and V H -V L ) were constructed ( Construction and expression of soluble C4 scFvs. scFvs frequently form inclusion bodies when they are overexpressed in bacterial cytoplasm (22,31,32). When pET-S tag vectors containing the C4 scFvs (Fig.…”

“…6A). Because the pectate lyase B (PelB) secretory signal peptide has been used for secretion of scFvs into the periplasm or medium to prevent aggregation in the cytoplasm (31,38,39), C4 scFv constructs carrying the PelB secretory signal peptide (PelB-V L -V H and PelB-V H -V L ) were created using a commercial pET20b(ϩ) vector (Novagen) to improve secretion. Unexpectedly, no bacterial colony growth was observed after the plasmid DNAs were transformed into E. coli strain BL21(DE3) or Rosetta (DE3) (data not shown).…”

“…The shorter production time and lower cost of scFvs compared with MAbs are also advantageous because they enable expression of scFvs in heterologous protein expression systems, such as Escherichia coli systems. However, scFvs can easily form insoluble aggregates when they are expressed in the E. coli cytoplasm (31,32). Therefore, the development of production methods for soluble scFv expression in bacteria would greatly enhance their applicability.…”

Indole generates quiescent and metabolically active Escherichia coli cultures

“…As a test of the synthetic capacity of Q-Cells, their ability to express a single-chain antibody fragment (scFv) in high-density shake flask and fermenter cultures was investigated [4]. The scFv gene was tagged with a pelB leader that resulted in the majority of product being secreted into the culture supernatant.…”