Single-domain antibody fusion proteins with the acid tail of alpha-synuclein
Inventors
Zabetakis, Daniel • Anderson, George P. • Goldman, Ellen R. • Turner, Kendrick • Brozozog Lee, P. Audrey
Assignees
Publication Number
US-10611809-B2
Publication Date
2020-04-07
Expiration Date
2034-01-17
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
A single-domain antibody (sdAb) is produced by causing a bacteria to express the sdAb into cytoplasm of the bacteria, wherein the sdAb is expressed as a fusion protein with the acid tail of α-synuclein. In embodiments, the protein is free of a periplasmic location tag. Such antibodies have the unexpected ability to refold after thermal denaturation.
Core Innovation
The invention relates to a fusion protein comprising a single-domain antibody (sdAb) and the acid tail of α-synuclein (ATS), expressed in the cytoplasm of bacteria without a periplasmic location tag. This fusion allows the sdAb to refold after thermal denaturation, overcoming limitations typically associated with cytoplasmic expression of sdAbs which lack disulfide bonds and thus have reduced melting temperatures and inability to refold after heating.
The problem addressed is that sdAbs, when expressed in the reducing cytoplasm of bacteria, do not refold after thermal denaturation, largely due to the absence of disulfide bond formation that normally stabilizes the protein. Periplasmic expression facilitates disulfide bond formation but yields lower protein production. There is a need for sdAbs with improved thermal refolding ability that can be produced at higher yields in the cytoplasm.
The solution disclosed is fusing the acid tail of α-synuclein to sdAbs, which unexpectedly promotes refolding after thermal denaturation even in the absence of disulfide bonds. While ATS has been known to stabilize proteins and prevent aggregation, its ability to promote refolding of sdAbs after heat denaturation was surprising. This fusion provides increased solubility and functional stability at high temperatures, enabling sdAbs that can be cytoplasmically produced with improved properties.
Claims Coverage
The patent includes two independent claims covering proteins comprising a single-domain antibody fused with the acid tail of α-synuclein, specifying features related to fusion and tagging.
Fusion protein of single-domain antibody and acid tail of α-synuclein free of periplasmic location tag
A fusion protein comprising an sdAb and the acid tail of α-synuclein, expressed without a periplasmic location tag to allow cytoplasmic expression and improved refolding ability.
Purified single-domain antibody joined to acid tail of α-synuclein
A purified protein comprising an sdAb joined to the acid tail of α-synuclein, enabling refolding post thermal denaturation and improved stability.
These features define fusion proteins of sdAbs linked with the acid tail of α-synuclein that lack periplasmic localization tags and are purified as single entities, enabling enhanced thermal refolding and stability when expressed cytoplasmically.
Stated Advantages
The fusion with the acid tail promotes refolding of sdAbs after thermal denaturation, increasing their functional stability.
Cytoplasmic expression of sdAb-ATS fusion proteins results in higher yields compared to periplasmic expression.
The fusion proteins exhibit increased solubility after heating, reducing aggregation compared to sdAbs without ATS.
Documented Applications
Use of sdAb-ATS fusion proteins in diagnostic and analytical kits and devices usable in field conditions without refrigeration.
Potential use of the fusion proteins as intrabodies that function inside cells without reliance on disulfide bonds, relevant for protection against intracellular toxins and viral inhibitors.
Interested in licensing this patent?