Cloning, expression and characterisation of a single-chain Fv antibody fragment against domoic acid in Escherichia coli

Hu, Xuejun; O’Dwyer, Ronan; Wall, J. Gerard

doi:10.1016/j.jbiotec.2005.05.018

Cited by 28 publications

(35 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Without any optimization, the soluble yield was satisfactory and very reproducible, and it is likely that yields could be improved considerably by coexpression of molecular chaperones (28) or the E. coli disulfide isomerase enzyme (15). The resulting soluble scFv gave a reproducible standard curve within the range required for enforcement of the regulatory cutoff level for DA in shellfish (250 mg/kg) (8) and could be used for the detection of DA in shellfish extracts.…”

Section: Discussionmentioning

confidence: 99%

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of thePseudonitzschia pungensToxin Domoic Acid

Finlay

Shaw

Reilly

et al. 2006

Appl Environ Microbiol

View full text Add to dashboard Cite

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V H and V L genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.The accurate quantification of algal toxins in environmental and food samples is critical for consumer protection. The levels of these low-molecular-weight toxins are typically measured using either bioassay or high-performance liquid chromatography methods (16, 23), both of which are time-consuming, relatively low-throughput, and often expensive procedures (11, 13). Immunoassays, by contrast, are more suited to highthroughput screening, while being sensitive and highly specific (30), and recent changes in European Union regulations governing the sale of shellfish (8) indicate that immunoassays are becoming more acceptable for shellfish monitoring programs.Immunoassay techniques have previously relied on hyperimmune polyclonal sera from rabbits, sheep, or other mammals. These are relatively easy to produce in large quantities but do not offer the consistency required for large-scale application or commercial production. More recently, monoclonal antibodies have been favored because they can be produced in unlimited supply and are more easily standardized. However, monoclonal antibodies have their own set of difficulties, as they are almost exclusively murine in origin (10) and are laborious to screen, and small mammals such as mice do not always provide the high-affinity antibody response to small hapten molecules needed for sensitive assay development (22).The limitations of traditional techniques have led several research groups to investigate the use of phage display to produce antihapten antibodies. The phage display of recombinant antibody libraries is a robust monoclonal antibody technology that is an increasingly attractive method, as it allows the ...

show abstract

Section: Discussionmentioning

confidence: 99%

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of thePseudonitzschia pungensToxin Domoic Acid

Finlay

Shaw

Reilly

et al. 2006

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Pellets were solubilized in 8 M urea in PBS, and soluble and insoluble fractions were analyzed by SDS-PAGE and Western blotting with an anti-M2 FLAG antibody. The binding of scFv molecules was tested by enzyme-linked immunosorbent assay (ELISA) according to published procedures (7). Briefly, wells of a 96-well microtiter plate were coated with 10 g of domoic acid (Calbiochem, United Kingdom)/ml conjugated to ovalbumin, followed by blocking with 5% milk powder in PBS.…”

mentioning

confidence: 99%

Trigger Factor from the Psychrophilic Bacterium Psychrobacter frigidicola Is a Monomeric Chaperone

et al. 2009

Self Cite

View full text Add to dashboard Cite

In eubacteria, trigger factor (TF) is the first chaperone to interact with newly synthesized polypeptides and assist their folding as they emerge from the ribosome. We report the first characterization of a TF from a psychrophilic organism. TF from Psychrobacter frigidicola (TF Pf ) was cloned, produced in Escherichia coli, and purified. Strikingly, cross-linking and fluorescence anisotropy analyses revealed it to exist in solution as a monomer, unlike the well-characterized, dimeric E. coli TF (TF Ec ). Moreover, TF Pf did not exhibit the downturn in reactivation of unfolded GAPDH (glyceraldehyde-3-phosphate dehydrogenase) that is observed with its E. coli counterpart, even at high TF/GAPDH molar ratios and revealed dramatically reduced retardation of membrane translocation by a model recombinant protein compared to the E. coli chaperone. TF Pf was also significantly more effective than TF Ec at increasing the yield of soluble and functional recombinant protein in a cell-free protein synthesis system, indicating that it is not dependent on downstream systems for its chaperoning activity. We propose that TF Pf differs from TF Ec in its quaternary structure and chaperone activity, and we discuss the potential significance of these differences in its native environment.The folding of cytosolic proteins is coordinated by three chaperone systems in eubacteria: trigger factor (TF), DnaK and its cofactors DnaJ and GrpE, and GroEL, together with its cofactor GroES. As TF alone interacts with the ribosomes, it is the first chaperone to bind newly synthesized polypeptides and assist their folding, whereupon it passes them to downstream chaperone systems (12). It has recently been shown that, for multidomain proteins, TF and the DnaK system cooperate to slow down folding and cause a shift to a posttranslational folding mode (24). TF also plays an important role in the regulation of protein translocation due to its position at the ribosome exit tunnel (1, 24).TF is highly abundant in E. coli (up to 40 M), with most TF molecules existing in an equilibrium between its monomeric and dimeric states in the cytosol (28). The high cytosolic concentration of TF has recently been linked to a distinct functional role of the chaperone in maintaining newly translated polypeptides in a folding-competent state in the E. coli cytoplasm (35). A distinct antichaperone activity has also been described for TF, however, whereby substoichiometric concentrations of TF lead to increased polypeptide aggregation, whereas high TF/polypeptide ratios can also delay folding as the chaperone maintains polypeptides in a non-native state without promoting their complete refolding (9).E. coli TF (TF Ec ) is composed of an N-terminal ribosomebinding tail (domain I), an internal domain with peptidyl-prolyl cis/trans isomerase (PPIase) activity (domain II), and a Cterminal domain III that is involved in its chaperone activity (26). The importance of the PPIase domain remains unclear as TF binds non-native nascent polypeptides lacking proline residues (29), w...

show abstract

“…One of the most important factors to influence the expression level of single chain antibody is the direction of the variable heavy chain and light chain in both yeast and the E. coli expression system (Hu et al 2005). V H -V L demonstrated better expression than V L -V H in this study (data not shown).…”

Section: Resultsmentioning

confidence: 59%

“…V H -V L demonstrated better expression than V L -V H in this study (data not shown). However, previous studies indicated that there is no consistent rule on the effect of direction, but rather it depends on the genes (Hu et al 2005).…”

Section: Resultsmentioning

confidence: 95%

Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris

Chang¹,

Choi²,

Chun³

2008

Biotechnol Lett

View full text Add to dashboard Cite

A synthetic gene coding for single-chain variable domain fragment antibody against mycotoxin zearalenone (scFv-ZEN) has been designed, constructed and expressed in Pichia pastoris. The native scFv-ZEN sequence was optimized to Pichia preference codon usage. The expression level of codon-optimized scFv-ZEN was slightly higher than that of native scFv-ZEN, and its maximum yield reached 328 mg total protein/l in flask culture. The binding activities of two selected clones to ZEN using surface plasmon resonance analysis were comparable or better than that of monoclonal antibody. Our results demonstrate the potential of soluble scFv-ZEN for developing a rapid and affordable immunoassay for detection of ZEN in food and feedstuff.

show abstract

Cloning, expression and characterisation of a single-chain Fv antibody fragment against domoic acid in Escherichia coli

Cited by 28 publications

References 22 publications

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of thePseudonitzschia pungensToxin Domoic Acid

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of thePseudonitzschia pungensToxin Domoic Acid

Trigger Factor from the Psychrophilic Bacterium Psychrobacter frigidicola Is a Monomeric Chaperone

Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris

Contact Info

Product

Resources

About