Charged peptide appendage to facilitate oriented protein covalent immobilization

Inventors

Anderson, George P. • Liu, Jinny Lin • Raphael, Marc P. • Goldman, Ellen R.

Assignees

US Department of Navy

Abstract

Genetic fusions of proteins, for example single-domain antibodies (sdAbs), with a positively-charged domain enhanced immobilization of active protein in a desired orientation.

Core Innovation

The invention describes a technique for oriented immobilization of recombinant proteins, especially single-domain antibodies (sdAbs), on surfaces using genetically fused positively charged peptide domains. These charged peptides are appended to either the N-terminus or C-terminus of the polypeptide and enable electrostatic attraction and covalent attachment to oppositely charged surfaces. This approach facilitates orientation of proteins such that their active or binding sites remain accessible, thereby enhancing activity upon immobilization and avoiding functional impairment during chemical labeling.

The problem addressed is the difficulty in achieving controlled orientation of proteins, especially antibodies and recombinant antibody fragments, upon covalent immobilization to surfaces. Conventionally, immobilization chemistries like carbodiimide or succinimide ester linkers link random carboxyl and amine residues distributed across the protein surface, resulting in variable and often suboptimal orientation that impairs binding activity. Prior methods either require specialized surfaces or non-natural amino acids. Thus, there is a need for a simple, generally applicable method to control protein orientation during covalent attachment, preserving maximum binding capacity.

The invention thus provides fusion proteins where a positively charged domain, such as sequences with high lysine content or positively charged leucine zippers, is genetically fused to antibodies or other proteins. This charged appendage interacts with a carboxyl-functionalized surface under common coupling chemistries (e.g., EDC/NHS), resulting in preferential attachment via the charged tail and oriented display of the functional domain away from the surface. Additionally, the charged tail serves as a highly accessible site for chemical conjugation of molecules like biotin or fluorophores, improving labeling efficiency and preserving function.

Claims Coverage

The claims focus on fusion proteins combining single-domain antibodies with a positively charged domain fused to the C-terminus of the antibody. There is one independent claim articulating this fusion construct.

The patent's claims cover fusion proteins where a single-domain antibody is genetically linked at the C-terminus to a specified positively charged peptide sequence, enabling oriented immobilization and enhanced functional activity.

Stated Advantages

Documented Applications