Negative tail fusions can improve ruggedness of single domain antibodies.
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.
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA.
Ellen R
ER
Goldman
NOVA Research Inc., 1900 Elkin St., Suite 230, Alexandria, VA 22308, USA.
P Audrey
PA
Brozozog-Lee
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA.
Dan
D
Zabetakis
Science and Engineering Apprenticeship Program, American Society for Engineering Education, 4555 Overlook Ave. SW, Washington, DC 20375, USA.
Kendrick B
KB
Turner
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA.
Scott A
SA
Walper
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA.
Jinny L
JL
Liu
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA. Electronic address: george.anderson@nrl.navy.mil.
George P
GP
Anderson
D004220:Disulfides; D004926:Escherichia coli; D058849:Protein Refolding; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004220:Disulfides; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D051844:alpha-Synuclein
Acid tail of α-synuclein; Aggregation; Refold; Solubility; Thermal stability