Nucleic acids for single-domain antibodies with thermal stability under cytoplasmic expression
Inventors
Zabetakis, Daniel • Anderson, George P. • Goldman, Ellen R. • Turner, Kendrick • Brozozog Lee, P. Audrey
Assignees
Publication Number
US-10577401-B2
Publication Date
2020-03-03
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 nucleic acid encodes 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 nucleic acids encoding fusion proteins comprising a single-domain antibody (sdAb) fused with the acid tail of α-synuclein (ATS). These fusion proteins are produced by causing bacteria to express the sdAb into the cytoplasm, notably without a periplasmic location tag. Such sdAb-ATS fusion proteins have the unexpected ability to refold after thermal denaturation, providing improved thermal stability.
The problem addressed by the invention arises from limitations in cytoplasmic expression of sdAbs in bacteria. Normally, sdAbs contain a single disulfide bond that contributes to thermal stability, but the cytoplasm is a reducing environment that prevents disulfide bond formation. As a result, cytoplasmically expressed sdAbs have a reduced melting temperature and do not refold after thermal denaturation, limiting their utility. Periplasmic expression avoids this but has lower yields due to export bottlenecks.
The invention overcomes these issues by fusing sdAbs to the acid tail of α-synuclein, a small heat shock protein known to prevent aggregation and protect proteins from damage. While previously ATS failed to promote refolding of enzymes after thermal denaturation, the present invention demonstrates that fusion with ATS significantly assists sdAb refolding. This enhancement allows production of functional, thermally stable sdAbs in the cytoplasm, increasing yields and enabling new commercial uses.
Claims Coverage
The patent contains two independent claims directed to nucleic acids encoding fusion proteins comprising a single-domain antibody and the acid tail of α-synuclein without a periplasmic tag. The main inventive features relate to the composition of the encoded fusion protein and expression characteristics.
Fusion protein composition
A nucleic acid encoding a fusion protein comprising a single-domain antibody (sdAb) and the acid tail of α-synuclein, wherein the fusion protein is free of a periplasmic location tag.
Protein encoding of sdAb joined to the acid tail of α-synuclein
A nucleic acid encoding a protein comprising a single-domain antibody joined to the acid tail of α-synuclein, specifying the fusion protein composition without requiring a periplasmic location tag.
The independent claims cover nucleic acids encoding fusion proteins of sdAbs fused with the acid tail of α-synuclein expressed without periplasmic localization tags, highlighting the fusion protein construct and its genetic encoding as the core inventive aspects.
Stated Advantages
The fusion of sdAbs with the acid tail of α-synuclein allows cytoplasmic expression of sdAbs that are capable of refolding after thermal denaturation, overcoming limitations caused by the reducing cytoplasmic environment.
The approach improves solubility of sdAbs after heating and reduces aggregation during thermal stress.
The fusion proteins retain binding affinity and function after exposure to elevated temperatures, contributing to enhanced thermal stability.
The method potentially increases protein yields by enabling production in the cytoplasm rather than the periplasm, avoiding export bottlenecks.
Documented Applications
Production of single-domain antibodies with improved thermal stability and refolding capability for diagnostic and/or analytical kits and devices usable in field conditions without refrigeration.
Intrabodies functioning inside cells to bind target molecules where sdAbs need to operate in the absence of their disulfide bonds, including protection from intracellular toxins and viral inhibitors.
Interested in licensing this patent?