Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

Song, Houyan; Jang, Jun-Hyuck; Kim, Young-Wan; Kim, Dong‐Hyung; Park, Sung-Goo; Lee, Myung Kyu; Paek, Se‐Hwan; Woo, Eui-Jeon

doi:10.3390/ijms151223658

Cited by 14 publications

(15 citation statements)

References 38 publications

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“…The far‐UV CD spectra (190–240 nm) for the six scFvs (Fig. d) showed the scFvs were predominantly β‐sheets, as expected for typical members of the immunoglobulin family, including scFvs (Supplementary Table SVI) (Blanco‐Toribio et al, ; Glaven et al, ; Gregoire et al, ; Lee et al, ; Song et al, ). The scFv‐2D10 (PDB ID‐ 4H0H) crystal structure showed the presence of 4% helical and 47% beta sheet secondary structures (Tapryal et al, ), whereas the CD data for the scFv‐2D10 in our hands retrieved 3.4, 5, and 9% helical and 40, 38, and 27% beta sheet structures from CDSSTR, CONTIN, and GOR4, respectively (Supplementary Table SVI).…”

Section: Resultssupporting

confidence: 59%

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Computational de novo design of antibodies binding to a peptide with high affinity

Poosarla¹,

Li²,

Goh

et al. 2017

Biotech & Bioengineering

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Antibody drugs play a critical role in infectious diseases, cancer, autoimmune diseases, and inflammation. However, experimental methods for the generation of therapeutic antibodies such as using immunized mice or directed evolution remain time consuming and cannot target a specific antigen epitope. Here, we describe the application of a computational framework called OptMAVEn combined with molecular dynamics to de novo design antibodies. Our reference system is antibody 2D10, a single-chain antibody (scFv) that recognizes the dodecapeptide DVFYPYPYASGS, a peptide mimic of mannose-containing carbohydrates. Five de novo designed scFvs sharing less than 75% sequence similarity to all existing natural antibody sequences were generated using OptMAVEn and their binding to the dodecapeptide was experimentally characterized by biolayer interferometry and isothermal titration calorimetry. Among them, three scFvs show binding affinity to the dodecapeptide at the nM level. Critically, these de novo designed scFvs exhibit considerably diverse modeled binding modes with the dodecapeptide. The results demonstrate the potential of OptMAVEn for the de novo design of thermally and conformationally stable antibodies with high binding affinity to antigens and encourage the targeting of other antigen targets in the future.

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Section: Resultssupporting

confidence: 59%

“…To confirm correct folding of scFvs, the secondary structures of the six purified scFvs were monitored by far‐UV CD spectroscopy and compared to that of other scFvs (Blanco‐Toribio et al, ; Glaven et al, ; Song et al, ). The far‐UV CD spectra (190–240 nm) for the six scFvs (Fig.…”

Section: Resultsmentioning

confidence: 99%

Computational de novo design of antibodies binding to a peptide with high affinity

Poosarla¹,

Li²,

Goh

et al. 2017

Biotech & Bioengineering

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“…One of the main drawbacks of E . coli as a host organism for protein expression and purification is due to protein expression in the insoluble fraction, as is the case for most scFvs in the literature [ 25 , 40 – 43 ] and also for scFv-h3D6-Ec. In this case, the insoluble fraction needs to be chemically solubilized and proteins, which are denatured, must be refolded.…”

Section: Resultsmentioning

confidence: 99%

Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect

et al. 2017

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ScFv-h3D6 has been shown as an efficient therapy in the 3xTg-AD mouse model of Alzheimer’s Disease. Because one of the major bottlenecks for the therapeutic uses of proteins produced in Escherichia coli is their potential contamination with endotoxins, LPS were extensively removed by a rather low-efficient, expensive, and time-consuming purification step. In addition, disulfide scrambling is favored in the reducing bacterial cytoplasm albeit the use of reductase deficient strains. To overcome these hurdles, as well as to improve the yield, the yeast Pichia pastoris, an endotoxin-free host system for recombinant protein production, has been used to produce scFv-h3D6, both in flask and in a fed-batch bioreactor. Comparison of the thermal stability of the obtained protein with that from E. coli showed no differences. Opposite to the case of the protein obtained from E. coli, no disulfide scrambled conformations or LPS traces were detected in that produced in P. pastoris. Cytotoxicity assays in SH-SY5Y neuroblastoma cell-cultures demonstrated that proteins from both expression systems were similarly efficient in precluding Aβ-induced toxicity. Finally, the 3xTg-AD mouse model was used to test the therapeutic effect of both proteins. Quantification of Aβ levels from cortex and hippocampus protein extracts by ELISA, and Aβ-immunohistochemistry, showed that both proteins reduced Aβ burden. This work demonstrates that scFv-h3D6 obtained from P. pastoris shows the same benefits as those already known for that obtained from E. coli, with multiple advantages in terms of recombinant production and safety.

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“…The production of scFvs in soluble and active form in E . coli is difficult to obtain, since the reducing environment of the bacterial cytoplasm is not compatible with disulfide bonds formation, resulting in unstable molecules or even aggregates [ 47 ], moreover several studies have reported the same issue with antibody fragments cloned into pET28a vector [ 45 , 48 , 49 , 50 ].…”

Section: Discussionmentioning

confidence: 99%

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

et al. 2015

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BackgroundDiarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains.Methods and FindingsRecombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.ConclusionThe developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

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Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

Cited by 14 publications

References 38 publications

Computational de novo design of antibodies binding to a peptide with high affinity

Computational de novo design of antibodies binding to a peptide with high affinity

Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

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