Isolation and Characterization of a Thermally Stable Recombinant Anti-Caffeine Heavy-Chain Antibody Fragment

Ladenson, Ruth C.; Crimmins, Dan L.; Landt, Yvonne; Ladenson, Jack H.

doi:10.1021/ac058044j

Cited by 124 publications

(110 citation statements)

References 18 publications

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“…Camelid VHH is famous for its high thermodynamic stability (25)(26)(27). Unique applications are proposed to utilize this high stability of VHH, such as detection of caffeine in hot beverages (28) and prevention of dandruff by VHH mixed with shampoo containing a high concentration of surfactant (29). Stabilization by the engineered disulfide bond reinforces the utility of VHHs.…”

Section: Discussionmentioning

confidence: 99%

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

Hagihara

Mine

Uegaki

2007

Journal of Biological Chemistry

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We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala 49 and Ile 70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10°C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala 49 and Ile 70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.One of the major objectives of antibody engineering is to stabilize the three-dimensional structure of the antibody. Disulfide bonds often significantly stabilize the structure of native proteins. Thus, the introduction of artificial disulfide bonds is recognized as a useful protein engineering technique to increase conformational stability. Although this technique has been applied to many proteins, there are no reports of engineered disulfide bonds in an immunoglobulin fold framework.Cystine is hydrophobic, and thus, most of naturally occurring disulfide bonds are buried in the protein (1-4). Therefore, the introduction of an engineered disulfide bond into the hydrophobic core better maintains the biophysical properties of the target protein. There are several examples of artificial disulfide bonds that can replace a pair of buried hydrophobic residues, the accessible surface areas of which were Ͻ20% (5-11). The introduction of a disulfide bond into the buried hydrophobic core of human carbonic anhydrase (A23C/L203C) markedly stabilizes this enzyme; the midpoint temperature of thermal unfolding (T m ) of the mutant is 10°C higher than that of the wild-type protein (6). The engineered disulfide bonds in alkaline protease AprP (G199C/F236C) (11), xylanase (V98C/ A152C) (7), and manganese peroxidase (A48C/A63C) (10) mutants increase their tolerance against heat inactivation. On the other hand, subtilisin BPNЈ mutants (V26C/A232C and A29C/M119C) exhibit similar or slightly lower stability to irreversible thermal inactivation (8). Tolerance against heat denaturation is not directly correlated with conformational stability. Only the mutational effect on human carbonic anhydrase II (6) was examined in a reversible system. Little information is available about the thermodynamic effe...

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Section: Discussionmentioning

confidence: 99%

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

Hagihara

Mine

Uegaki

2007

Journal of Biological Chemistry

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“…The binding site formed from a single domain that is small, highly stable, and able to refold properly after denaturation, makes the HcAb variable domain a valuable source of alternative recombinant binding ligand for many applications. [24][25][26] The aim of this research was to evaluate the diphtheria toxin-binding capability of antibodies obtained from the immunized camel. Earlier work had shown that camel HcAb was active towards pathogens, and the ability to create single-domain antibodies from immunized libraries attests to their antigen recognition capability.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of antidiphtheria toxin nanobodies

Shaker¹

2010

NSA

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Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Conventional antibodies are glycoproteins comprising two heavy and two light chains. Surprisingly, all members of the Camelidae family possess a fraction of antibodies devoid of both light chains and the first constant domain. These types of antibodies are known as heavy-chain antibody (HcAb) nanobodies. There are three subclasses of IgG in dromedaries, namely IgG1, IgG2, and IgG3 of which IgG2 and IgG3 are of the HcAb type. These heavy chain antibodies constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. In the present work, the different IgG subclasses from an immunized camel (Camelus dromedarius) with divalent diphtheria-tetanus vaccine were purified using their different affinity for protein A and protein G and their absorbance measured at 280 nm. Purity control and characterization by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of IgG subclasses was done under reducing conditions. Protein bands were visualized after staining with Coomassie Blue, showing two bands at 50 kDa and 30 kDa for IgG1, while IgG2 and IgG3 produced only one band at 46 kDa and 43 kDa, respectively. An enzyme-linked immunosorbent assay test using diphtheria toxin and purified IgG subclasses from the immunized camel were performed to evaluate their efficiency. Compared with conventional IgG1, heavy chain antibodies (nanobodies) were shown to be more efficient in binding to diphtheria toxin antigen. This study revealed the possibility of using IgG2 and IgG3 nanobodies as an effective antitoxin for the treatment of diphtheria in humans.

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“…Several anticaffeine VHHs have been already generated for the quantification of caffeine in hot beverages. At 70°C, one of these VHHs could recognize caffeine and amazingly recover its binding functionality after an incubation step at 90°C, 110 demonstrating the excellent thermostability of these nanobodies.…”

Section: Nanobodies For Agricultural Protection and Food Analysismentioning

confidence: 95%

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

Wang

Fan

Shao

et al. 2016

IJN

126

107

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Owing to peculiar properties of nanobody, including nanoscale size, robust structure, stable and soluble behaviors in aqueous solution, reversible refolding, high affinity and specificity for only one cognate target, superior cryptic cleft accessibility, and deep tissue penetration, as well as a sustainable source, it has been an ideal research tool for the development of sophisticated nanobiotechnologies. Currently, the nanobody has been evolved into versatile research and application tool kits for diverse biomedical and biotechnology applications. Various nanobody-derived formats, including the nanobody itself, the radionuclide or fluorescent-labeled nanobodies, nanobody homo- or heteromultimers, nanobody-coated nanoparticles, and nanobody-displayed bacteriophages, have been successfully demonstrated as powerful nanobiotechnological tool kits for basic biomedical research, targeting drug delivery and therapy, disease diagnosis, bioimaging, and agricultural and plant protection. These applications indicate a special advantage of these nanobody-derived technologies, already surpassing the “me-too” products of other equivalent binders, such as the full-length antibodies, single-chain variable fragments, antigen-binding fragments, targeting peptides, and DNA-based aptamers. In this review, we summarize the current state of the art in nanobody research, focusing on the nanobody structural features, nanobody production approach, nanobody-derived nanobiotechnology tool kits, and the potentially diverse applications in biomedicine and biotechnology. The future trends, challenges, and limitations of the nanobody-derived nanobiotechnology tool kits are also discussed.

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Isolation and Characterization of a Thermally Stable Recombinant Anti-Caffeine Heavy-Chain Antibody Fragment

Cited by 124 publications

References 18 publications

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

Evaluation of antidiphtheria toxin nanobodies

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

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