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Negative tail fusions can improve ruggedness of single domain antibodies

Ellen R Goldman1, P Audrey Brozozog-Lee2, Dan Zabetakis1

  • 1Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA.

Protein Expression and Purification
|January 21, 2014
PubMed

Single-domain antibodies (sdAbs), the recombinantly expressed binding domains derived from the heavy-chain-only antibodies found in camelids and sharks, are valued for their ability to refold after heat denaturation. However, some sdAbs are prone to aggregation on extended heating at high concentration. Additionally, sdAbs prepared cytoplasmically often lack the conserved disulfide bond found in variable heavy domains, which both decreases their melting point and can decrease their ability to refold. Genetic fusions of sdAbs with the acid tail of α-synuclein (ATS) resulted in constructs that had enhanced ability to resist aggregation. In addition, almost complete refolding was observed even in the absence of the disulfide bond. These sdAb-ATS fusions expand the utility of sdAbs. They provide sdAbs that are resistant to aggregation, and enable the production of re-foldable sdAbs in the reducing environment of the cytoplasm.

Keywords:
Acid tail of α-synucleinAggregationRefoldSolubilityThermal stability

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