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

2004 ◽  
Vol 70 (12) ◽  
pp. 7033-7039 ◽  
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.

scholarly journals A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants

1999 ◽  
Vol 262 (2) ◽  
pp. 617-624 ◽  
Author(s):  
Paraskevi Tavladoraki ◽  
Alessandra Girotti ◽  
Marcello Donini ◽  
Francisco Javier Arias ◽  
Camillo Mancini ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transgenic Plants ◽  
Antibody Fragment ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Single Chain Antibody Fragment

scholarly journals Studies of Single-Chain Antibody Expression in Quiescent Escherichia coli

2004 ◽  
Vol 70 (5) ◽  
pp. 3005-3012 ◽  
Author(s):  
K. J. Mukherjee ◽  
D. C. D. Rowe ◽  
N. A. Watkins ◽  
D. K. Summers
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Formation Rate ◽  
Antibody Fragment ◽  
Control Culture ◽  
Temperature Sensitive ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Specific Product ◽  
Quiescent Cells

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.

scholarly journals Engineering of The Bacillus Subtilis PhoD Signal Peptide To Direct High-Level, Tat-Specific Export of A Single-Chain Antibody Fragment To The Periplasm in Eschericha Coli

2020 ◽  
Author(s):  
Chillel Jawara ◽  
Kirsty L Richards ◽  
Amber R Peswani ◽  
Kelly L Walker ◽  
Lara Nascimento ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Antibody Fragment ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
E Coli ◽  
Tat Pathway ◽  
Single Chain Antibody Fragment

Abstract Background: Numerous high-value proteins have been produced in E. coli, and a favoured strategy is to export the protein of interest to the periplasm by means of an N-terminal signal peptide. While the Sec pathway has been extensively used for this purpose, the Tat pathway has potential because it transports fully-folded heterologous proteins. Most studies on the Tat pathway have used the E. coli TorA signal peptide to direct export, because it is highly Tat-specific, unlike many Tat signal peptides which can also function as Sec signal peptides. However, the TorA signal peptide is prone to degradation in the cytoplasm, leading to reduced export rates in some cases. Here, we have tested a range of alternative signal peptides for their ability to direct Tat-dependent export of a single-chain antibody fragment (scFv). Results: We show that the signal peptides of E. coli AmiC, MdoD and YcbK direct efficient export of the scFv by both the Tat and Sec pathways, which may be a disadvantage when Tat-specific export is required. The same applies to the Tat signal peptide of Bacillus subtilis PhoD, which likewise directs efficient export by Sec. We engineered the PhoD signal peptide by introduction of a Lys or Asn residue in the C-terminal domain of the signal peptide, and we show that this substitution renders the signal peptide Tat-specific. These signal peptides, designated PhoDk and PhoDn, direct efficient export of scFv in shake flask and fed-batch fermentation studies, reaching export levels that are well above those obtained with the TorA signal peptide. Culturing in ambr250 bioreactors was used to fine-tune the growth conditions, and the net result was export of the scFv by the Tat pathway at levels of approximately 1g protein/L culture. Conclusions: The new PhoDn and PhoDk signal peptides have significant potential for the export of heterologous proteins by the Tat system.

scholarly journals Cosecretion of Chaperones and Low-Molecular-Size Medium Additives Increases the Yield of Recombinant Disulfide-Bridged Proteins

2001 ◽  
Vol 67 (9) ◽  
pp. 3994-4000 ◽  
Author(s):  
Jörg Schäffner ◽  
Jeannette Winter ◽  
Rainer Rudolph ◽  
Elisabeth Schwarz
Keyword(s):  
Antibody Fragment ◽  
Molecular Size ◽  
Tissue Type ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
E Coli ◽  
Positive Effects ◽  
Type Plasminogen Activator ◽  
Model Protein

ABSTRACT Attempts were made to engineer the periplasm of Escherichia coli to an expression compartment of heterologous proteins in their native conformation. As a first approach the low-molecular-size additive l-arginine and the redox compound glutathione (GSH) were added to the culture medium. Addition of 0.4 Ml-arginine and 5 mM reduced GSH increased the yield of a native tissue-type plasminogen activator variant (rPA), consisting of the kringle-2 and the protease domain, and a single-chain antibody fragment (scFv) up to 10- and 37-fold, respectively. A variety of other medium additives also had positive effects on the yield of rPA. In a second set of experiments, the effects of cosecreted ATP-independent molecular chaperones on the yields of native therapeutic proteins were investigated. At optimized conditions, cosecretion of E. coli DnaJ or murine Hsp25 increased the yield of native rPA by a factor of 170 and 125, respectively. Cosecretion of DnaJ also dramatically increased the amount of a second model protein, native proinsulin, in the periplasm. The results of this study are anticipated to initiate a series of new approaches to increase the yields of native, disulfide-bridged, recombinant proteins in the periplasm ofE. coli.

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