Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

Oelschlaeger, Peter; Lange, Stefan; Schmitt, Jutta; Siemann, Martin; Reuß, Matthias; Schmid, Rolf D.

doi:10.1007/s00253-002-1190-6

Cited by 18 publications

(9 citation statements)

References 35 publications

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“…Out of all the recombinant systems studied, E. coli/pCS22 host was found to be optimum because of its efficient protein expression, folding and secretion to the periplasmic space. The total protein concentration in the periplasmic fraction (96 mg/L) was higher than in previous published reports on periplasmic protein expression using pelB leader sequence [23,24]. This variation in total recombinant protein concentration may be due to variation in vector characteristics, choice and source of gene or choice of host organism.…”

Section: Cell Growth and A-amylase Expression In Bioreactorcontrasting

confidence: 80%

Cloning and Expression ofα-Amylase Gene inEscherichia coli: Effect on Specific Oxygen Uptake Rate and Host Cell Morphology during Batch Fermentation

Mathur

Chand

2011

Indian Chemical Engineer

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Cloning and expression of any heterologous gene product always impart metabolic load on the recombinant host for utilization of its metabolites that results in variations in cellular physiology. An understanding of such stress is important for robust process design and scaleup. In the present communication, an attempt has been made to explain the metabolic load imposed on the recombinant strain of Escherichia coli BL21(DE3) owing to over-expression of amyE gene, using different expression vectors of varying size. Current studies show that metabolic quotient or specific oxygen uptake rate (SOUR) of different recombinant hosts increases with an increase in vector size, showing correlation between vector size and cellular metabolic load. Further increase in SOUR on protein expression shows even higher metabolic load for protein expression than plasmid maintenance. Increase in bacterial size and decrease in specific growth rate on transformation and recombinant protein expression are cellular responses to additional metabolic load.

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Section: Cell Growth and A-amylase Expression In Bioreactorcontrasting

confidence: 80%

Cloning and Expression ofα-Amylase Gene inEscherichia coli: Effect on Specific Oxygen Uptake Rate and Host Cell Morphology during Batch Fermentation

Mathur

Chand

2011

Indian Chemical Engineer

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“…Therefore, in E. coli it is usually desirable to express scFvs as fusion proteins targeted for translocation to the oxidative periplasm to obtain functional products (3,26). Various vector systems for recombinant scFv expression in E. coli have been reported (11,15,19), but the experiments were typically performed in shake-flask cultures with product yields of 10 to 30 mg/liter. Plasmid-based gene expression under high-cell-density cultivation may dramatically promote problems related to plasmid instability and lysis of recombinant cells (16,28).…”

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confidence: 99%

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

Sletta

Nedal

Aune

et al. 2004

Appl Environ Microbiol

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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.The application range of antibodies in medicine and biotechnology is broad, and there has been great progress in the design and selection of new variants with novel affinities. In particular, there has been a growing interest in the development of antibody fragments comprising the V H and V L domains connected to each other as a single chain (scFv) (4). The small size of scFv proteins (about 250 amino acids) compared to native antibodies confers certain therapeutic advantages because of their shorter half-life (rapid blood clearance) and faster tissue penetration. scFv molecules can be used as selective carriers for delivering radionucleids, toxins, or cytotoxic drugs to malignant cell populations, as well as providing valuable tools for studying antibody-antigen interactions in detail (14). In addition, this feature makes it possible to construct and screen large scFv libraries by using phage display approaches (for a review, see reference 23).For medical applications scFvs are needed in large amounts, and the ability to produce high yields in Escherichia coli has gained considerable interest (14). Native scFv proteins have two disulfide bonds and require oxidative conditions to fold correctly. Although expression of native scFv proteins without disulfide bridge formation has been reported (9), cytoplasmic production in bacteria typically results in aggregation of scFv polypeptides into insoluble inclusion bodies (14). Therefore, in E. coli it is usually desirable to express scFvs as fusion proteins targeted for translocation to the oxidative periplasm to obtain functional products (3,26). Various vector systems for recombinant scFv expression in E. coli have been reported (11,15,19), but the experiments were typically perfo...

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“…The Pel B leader sequence in the expression plasmid facilitated the transportation of the scFv into the oxidizing environment of the E. coli periplasmic space, a space where two intra-domain disulfide bonds in the scFv can take place. (52) However, previous findings have indicated that the presence of unpaired cysteine at the C-terminus affects the yield of soluble scFv production by forming covalent aggregates with other proteins containing cysteine within the periplasmic space. (10,32) To overcome this obstacle, the conditions for hscFv-C production were investigated.…”

Section: Discussionmentioning

confidence: 99%

Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application

Khantasup

Chantima

Sangma

et al. 2015

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

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Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

Cited by 18 publications

References 35 publications

Cloning and Expression ofα-Amylase Gene inEscherichia coli: Effect on Specific Oxygen Uptake Rate and Host Cell Morphology during Batch Fermentation

Cloning and Expression ofα-Amylase Gene inEscherichia coli: Effect on Specific Oxygen Uptake Rate and Host Cell Morphology during Batch Fermentation

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

Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application

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