Bivalent Fv Antibody Fragments Obtained by Substituting the Constant Domains of a Fab Fragment with Heterotetrameric Molybdopterin Synthase

Petrov, Kliment; Dion, Michel; Hoffmann, Lionel; Dintinger, Thierry; Defontaine, Alain; Tellier, Charles

doi:10.1016/j.jmb.2004.06.075

Cited by 13 publications

(15 citation statements)

References 33 publications

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“…Binding was measured by single injection at 20 ll/min. Biacore analysis indicated specific binding of the purified protein to HLA-A2-Tax [11][12][13][14][15][16][17][18][19] , but not to HLA-NY-ESO-1 157-165 (irrelevant peptide control). The number of response units observed correlated approximately to the volume of the initial culture (Fig.…”

Section: Surface Plasmon Resonancementioning

confidence: 99%

“…Biotinylated peptide-HLA-A*0201 complexes [34] (100 ll/well at 5 lg/ml) were coupled to the plate via streptavidin. The two peptide-HLA complexes used were HLA-A2-Tax [11][12][13][14][15][16][17][18][19] LLFGYVYV (A6 TCR epitope) and HLA-A2-NY-ESO-1 157-165 SLLMWITQC (irrelevant peptide, acts as a control for specific binding). Coated plates were blocked with 3% milk powder-phosphate buffered saline (w/v) (Marvel dried skimmed milk less than 1% fat) at room temperature for 2 h. Serial dilutions of both a control HLA-A2-Tax-specific TCR, A6c134 refolded from E. coli inclusion bodies (Fig.…”

Section: Elisamentioning

confidence: 99%

“…Surface plasmon resonance was used to test the activity of antibody Sepharose-purified A6c134 TCR (Table 2) by binding to HLA-A2-Tax [11][12][13][14][15][16][17][18][19] . Soluble fractions from a 50-ml and a 200-ml culture were prepared and separately purified on a Sepharose column.…”

Section: Surface Plasmon Resonancementioning

confidence: 99%

“…2). The amount of HLA-A2-Tax [11][12][13][14][15][16][17][18][19] specific A6c134 TCR in the load and eluted fractions from the HB9282 affinity column was determined by ELISA. Background (non-specific) binding was measured using HLA-A2-NY-ESO-1 157-165 .…”

Section: Surface Plasmon Resonancementioning

confidence: 99%

“…Combining trxB/gor mutations further enhances E. coli ability to form disulfide bonds in the cytoplasm [14]. This has been demonstrated by the successful expression of several Fab fragments in the cytoplasm of trxB gor mutant strains [19,20].…”

Section: Introductionmentioning

confidence: 98%

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Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of Escherichia coli trxB gor Mutants

et al. 2010

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Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed alpha and beta chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.

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Section: Surface Plasmon Resonancementioning

confidence: 99%

Section: Elisamentioning

confidence: 99%

Section: Surface Plasmon Resonancementioning

confidence: 99%

Section: Surface Plasmon Resonancementioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

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Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of Escherichia coli trxB gor Mutants

et al. 2010

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The History of Therapeutic Monoclonal Antibodies

Sodoyer

2016

Biosimilars of Monoclonal Antibodies

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Survey of the year 2004 commercial optical biosensor literature

Rich

Myszka

2005

J of Molecular Recognition

115

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The year 2004 represents a milestone for the biosensor research community: in this year, over 1000 articles were published describing experiments performed using commercially available systems. The 1038 papers we found represent a $ 10% increase over the past year and demonstrate that the implementation of biosensors continues to expand at a healthy pace. We evaluated the data presented in each paper and compiled a 'top 10' list. These 10 articles, which we recommend every biosensor user reads, describe wellperformed kinetic, equilibrium and qualitative/screening studies, provide comparisons between binding parameters obtained from different biosensor users, as well as from biosensor-and solution-based interaction analyses, and summarize the cutting-edge applications of the technology. We also re-iterate some of the experimental pitfalls that lead to sub-optimal data and over-interpreted results. We are hopeful that the biosensor community, by applying the hints we outline, will obtain data on a par with that presented in the 10 spotlighted articles. This will ensure that the scientific community at large can be confident in the data we report from optical biosensors.

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Bivalent Fv Antibody Fragments Obtained by Substituting the Constant Domains of a Fab Fragment with Heterotetrameric Molybdopterin Synthase

Cited by 13 publications

References 33 publications

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of Escherichia coli trxB gor Mutants

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of Escherichia coli trxB gor Mutants

The History of Therapeutic Monoclonal Antibodies

Survey of the year 2004 commercial optical biosensor literature

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