Improving the biophysical properties of anti-ricin single-domain antibodies

Turner, Kendrick B.; Liu, Jinny L.; Zabetakis, Dan; Lee, P. Audrey Brozozog; Anderson, George P.; Goldman, Ellen R.

doi:10.1016/j.btre.2015.01.001

Cited by 35 publications

(55 citation statements)

References 42 publications

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“…In addition, in contrast to the wildtype anti-hCG VHH, protein concentration dependencies of heat denaturation were observed in 2-SS, 1-SS and 0-SS (WA) mutants at >1 mM of protein concentration. Recently, Turner et al (38) reported the heat resistance of three different VHHs and their mutants with different charge distribution. Although the heat resistance of these VHHs was generally high, one of the wild-type VHHs was only marginally heat tolerant at 72 C. Mutations introducing more negatively charged amino acids than parental VHH improved the heat resistance.…”

Section: Discussionmentioning

confidence: 99%

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

2015

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Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heatinduced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability.Keywords: antibody engineering/disulfide bonds/protein chemical modification/protein engineering/protein stability.Abbreviations: bLA, Bacillus cereus b lactamase; CD, circular dichroism; hCG, human chorionic gonadotropin; SPR, surface plasmon resonance; t 1/2 90 C , halflife of VHH at 90 C; T m , mid-point temperature of thermal unfolding; VHH, variable domain of camelid heavy chain antibody.The variable domain of the camelid heavy chain antibody (VHH) is known to be highly heat resistant.VHHs remain active even after incubation at high temperatures (>80 C) because of their ability to refold to their native structure from a heat-induced unfolded state (1 6). However, even for VHHs, a long incubation finally leads to denaturation: e.g. the half-life of VHH at 90 C (t 1/2 90 C ) was 3 h at neutral pH (7). Residual activity of VHH depends only on the incubation time at 90 C, and not on the number of heating (90 C)-cooling (20 C) cycles, indicating that folding and unfolding intermediates are irrelevant to the loss of activity. Heat denaturation of VHHs is independent of protein concentration; therefore, VHHs lose their activity in a unimolecular manner, not by aggregation. This strongly suggests that chemical modifications play an important role during VHH denaturation. Indeed, replacing the Asn residues, which are prone to chemical modification, increase the heat resistance of VHHs (7).VHHs typically have only one intramolecular disulfide bond. The introduction of a second non-canonical artificial disulfide bond connecting the amino acids at positions 49 and 69, with numbering according to Kabat et al. (8), results in a significantly higher midpoint temperature of equilibrium thermal unfolding (T m ) in comparison to wild-type protein (9 13). In spite of its superior equilibrium thermodynamic stability, the mutant with a second disulfide bond exhibited lower residual activity ...

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Section: Discussionmentioning

confidence: 99%

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

2015

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“…Measuring the initial on-rate has the advantage that one does not require a standard curve and thus is a simple, sensitive, and direct measure of binding activity (31). …”

Section: Analytical Techniques For Studying Sdab Stabilitymentioning

confidence: 99%

Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview

et al. 2017

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Single domain antibodies (sdAbs) are gaining a reputation as superior recognition elements as they combine the advantages of the specificity and affinity found in conventional antibodies with high stability and solubility. Melting temperatures (Tms) of sdAbs cover a wide range from below 50 to over 80°C. Many sdAbs have been engineered to increase their Tm, making them stable until exposed to extreme temperatures. SdAbs derived from the variable heavy chains of camelid and shark heavy chain-only antibodies are termed VHH and VNAR, respectively, and generally exhibit some ability to refold and bind antigen after heat denaturation. This ability to refold varies from 0 to 100% and is a property dependent on both intrinsic factors of the sdAb and extrinsic conditions such as the sample buffer ionic strength, pH, and sdAb concentration. SdAbs have also been engineered to increase their solubility and refolding ability, which enable them to function even after exposure to temperatures that exceed their melting point. In addition, efforts to improve their stability at extreme pH and in the presence of chemical denaturants or proteases have been undertaken. Multiple routes have been employed to engineer sdAbs with these enhanced stabilities. The methods utilized to achieve these goals include grafting complementarity-determining regions onto stable frameworks, introduction of non-canonical disulfide bonds, random mutagenesis combined with stringent selection, point mutations such as inclusion of negative charges, and genetic fusions. Increases of up to 20°C have been realized, pushing the Tm of some sdAbs to over 90°C. Herein, we present an overview of the work done to stabilize sdAbs derived from camelids and sharks. Utilizing these various strategies sdAbs have been stabilized without significantly compromising their affinity, thereby providing superior reagents for detection, diagnostic, and therapeutic applications.

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“…The wild type sequences for all but two of the sdAb prepared here have been previously published in other works (Conway et al, 2010;Richard et al, 2013;Vance et al, 2013;Liu et al, 2015;Turner et al, 2015). The sequences as produced for this work with the location of the added cysteines that formed the additional disulfide bond marked are shown in Fig.…”

Section: Reagent and Buffersmentioning

confidence: 99%

“…The sdAb were produced as previously described (Turner et al, 2015). Briefly, the PET22b plasmid carrying the sdAb gene and the pTUM4 plasmid were transformed into Tuner (DE3) for expression and grown on agar plates containing both ampicillin and chloramphenicol (100 µg/mL and 34 µg/mL respectively).…”

Section: Recombinant Protein Production and Purificationmentioning

confidence: 99%

“…The 50 mL cultures were used to inoculate 0.5 L of TB (with appropriate antibiotics) which was grown for 2 hours at 25°C and then induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown a further 2 hours. Cultures were centrifuged and the cell pellets subject to an osmotic shock protocol (Turner et al, 2015). Protein was purified by Ni-NTA followed by size exclusion chromatography and was quantified by absorbance at 280 nm.…”

Section: Recombinant Protein Production and Purificationmentioning

confidence: 99%

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Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm

Shriver‐Lake

Goldman

Zabetakis

et al. 2017

Journal of Immunological Methods

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Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together β-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process. Here, we demonstrate that inclusion of the helper plasmid pTUM4, which results in the expression of four chaperones, DsbA, DsbC, FkpA, and SurA, increased yield on average 3.5-fold for the nine multi-disulfide bond single domain antibodies evaluated. No increase in production was observed for single domain antibodies containing only the canonical disulfide bond.

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Improving the biophysical properties of anti-ricin single-domain antibodies

Abstract: Graphical abstract

Cited by 35 publications

References 42 publications

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview

Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm

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