Metal chelate guided site-specific bioconjugation of polyhistidine-tagged proteins and native antibodies

Abstract

Bioconjugation is a field which has current real-world applications; however often these applications are reliant on lysine and cysteine conjugation methods using NHS esters and maleimide conjugation respectively. These methods offer little site-specificity which results in a two-fold issue of varying numbers of conjugates in varying locations between proteins. This work describes a novel linker that can be used for the process of site-specific bioconjugation. The linker consists of an NTA group; diazirine group and alkyne chain. The NTA group offers site-specific labelling in the presence of cobalt to histidine groups; the diazirine group forms a covalent bond to the protein after UV irradiation; the alkyne group allows for easy addition of azide containing functionalities. The linker has then been shown to successfully conjugate azide functionalities to a range of His-tagged proteins and a native histidine rich region of mouse IgG. For antibodies; the conjugation method was Fc specific; as shown by antibody reduction and papain digestion and allowed for a method for the covalent site-specific attachment of biotinylated antibodies for the purposes of oriented immobilisation onto streptavidin coated surfaces. Finally, the use of a Co3+ chelate via oxidation of Co2+ using H2O2 allows for the formation of an "Exchange-inert" chelate. This has been shown to successfully conjugate the commercially available NTA-biotin to native IgG in order for oriented immobilisation to a streptavidin coated 96-well plate for the purposes of ELISA assays.

Date of Award	27 Jul 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Lu Shin Wong (Main Supervisor) & Alex Jones (Co Supervisor)