Bioproduction and characterisation of recombinant human transcobalamin II for the development of a rapid cobalamin assay

Cobalamin (Cbl, vitamin B12) is an essential cofactor for two crucial Cbl-dependent enzymes impacting numerous metabolic activities, including DNA methylation. Holotranscobalamin (holoTC), a metabolically active form of Cbl, is one of the biomarkers for assessing Cbl status. However, current diagnostic tests rely on large, automated analysers, limiting access in point- of-care settings, especially in remote and low-income areas. This limited access is further compounded by the increasing global adoption of plant-based diets and ultra-processed foods, which, without Cbl supplementation, significantly increases the risk of Cbl deficiency and potentially severe, irreversible pathologies. This study focuses on the bioproduction and characterisation of human transcobalamin (TC) to develop a novel cobalamin assay. The primary aim was to produce recombinant TC protein in Escherichia coli to develop an ELISA-based assay for determining Cbl status in humans. Achieving stable and functional expression of human TC in E. coli is a significant advancement, as previous expression in prokaryotic systems resulted in non-functional TC incapable of binding cobalamin. In this study, we successfully expressed functional cobalamin-binding TC in E. coli using CyDisCo technology. This involved the co-expression of yeast Erv1p and human PDI to facilitate proper disulphide bond formation and isomerisation. Biophysical characterisation using circular dichroism, stopped-flow kinetics, and fluorimetry revealed that the recombinant TC exhibited similar properties to native TC reported in the literature, with a dissociation constant of 3.4 nM. We also successfully generated IgY antibodies raised in chickens against the recombinant TC for ELISA development. However, the resulting assay demonstrated a lower detection limit of 800 pM for holoTC, which is not sensitive enough to detect physiological levels of 19 - 140 pM. This work represents a crucial step towards developing a cobalamin point-of-care testing assay, although further optimisation is required to enhance its sensitivity for diagnostic application.