Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection

Walper, Scott A.; Battle, Shawna R.; Lee, P. Audrey Brozozog; Zabetakis, Dan; Turner, Kendrick B.; Buckley, Patricia E.; Calm, Alena M; Welsh, Heather; Warner, Candice R.; Zacharko, Melody; Goldman, Ellen R.; Anderson, George P.

doi:10.1016/j.ab.2013.10.031

Cited by 17 publications

(14 citation statements)

References 36 publications

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“…When VHHs are produced in the E. coli cytoplasm, which has a relatively reducing environment, the resultant VHHs are found to have a much lower T m than the same VHHs produced in the oxidizing environment of the E. coli periplasm (40–42). This difference in stability is attributable to the failure of the conserved disulfide bond to form.…”

Section: Stabilizing Vhhsmentioning

confidence: 99%

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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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Section: Stabilizing Vhhsmentioning

confidence: 99%

“…In one instance, we utilized the maltose-binding protein (MBP) isolated from the thermophile Pyrococcus furiosus to form a VHH-PfuMBP fusion (40). The primary purpose of this fusion was to facilitate production of a VHH in the E. coli cytoplasm at higher levels than achievable from the periplasm.…”

Section: Stabilizing Vhhsmentioning

confidence: 99%

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

et al. 2017

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“…As aggregation is also a problem with scFvs, recombinant binding domains derived from conventional antibodies, several strategies have been reported that led to more soluble and stable recombinant antibody-binding domains (rAbs). One successful strategy has been to produce sdAbs as fusions with a thermostable protein, as demonstrated by a fusion between a sdAb and a thermostable maltose binding protein that was able to withstand heating to 70 °C for an hour without loss of activity [10]. Increasing the net charge of the protein also can lead to more soluble rAbs.…”

Section: Introductionmentioning

confidence: 99%

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

Turner

Liu

Zabetakis

et al. 2015

Biotechnology Reports

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“…The isolation of 0.52 mg soluble MBP‐IW fusion protein from 1 g E. coli wet cell pellet appears to be low when compared to other studies describing the overexpression of MBP‐fusion proteins . It should be noted, however, that in these studies natural proteins were expressed while the IW precursor protein described here represents a repetitive artificial construct exhibiting a primary structure that does not occur in living systems.…”

Section: Discussionmentioning

confidence: 74%

Biotechnological production of the angiotensin‐converting enzyme inhibitory dipeptide isoleucine‐tryptophan

Michelke

Deußen

Kettner

et al. 2018

Engineering in Life Sciences

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Peptides with angiotensin‐converting enzyme (ACE)‐inhibitory and antihypertensive effects are suggested as innovative food additives to prevent or treat hypertension. Currently, these substances are isolated from food proteins following nonselective hydrolysis as a mixture of ACE‐inhibitory peptides and other protein fragments. This study presents an innovative biotechnological method, based on recombinant DNA technology that was established to specifically produce the ACE‐inhibitory dipeptide isoleucine‐tryptophan. In a first step, a repetitive isoleucine‐tryptophan construct fused to the maltose‐binding protein was generated and expressed in Escherichia coli BL21 cells. The chromatographically purified recombinant fusion protein was enzymatically hydrolyzed using α‐chymotrypsin to liberate the dipeptide isoleucine‐tryptophan. The identity of the liberated isoleucine‐tryptophan was confirmed by MS and derivatization of its N‐terminus. The ACE‐inhibitory effect of the recombinant dipeptide on soluble and membrane bound ACE was found to be indistinguishable from the inhibitory potential of the chemically produced commercially available dipeptide. The established experimental strategy represents a promising approach to the biotechnical production of sufficient amounts of recombinant peptide‐based ACE‐inhibitory and antihypertensive substances that are applicable as functional food additives to delay or even prevent hypertension.

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Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection

Cited by 17 publications

References 36 publications

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

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

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

Biotechnological production of the angiotensin‐converting enzyme inhibitory dipeptide isoleucine‐tryptophan

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