%0 Journal Article
%T Expression, Sarkosyl Solubilization, DNase Activity, Purification, and SPR Binding Affinity of Recombinant Diphtheria Toxoid (rCRM197EK) Expressed in Escherichia coli BL21(DE3).
%A Novianti MT
%A Subroto T
%A Efendi YS
%A Baroroh U
%A Kusumawardani S
%A Gumilar G
%A Yusuf M
%A Gaffar S
%J Mol Biotechnol
%V 0
%N 0
%D 2024 Aug 6
%M 39107502
%F 2.86
%R 10.1007/s12033-024-01238-y
%X CRM197EK is a derivative of diphtheria toxoid cross-reactive material-197 (CRM197) with two-point mutations (K51E and E148K) to improve its properties for a vaccine conjugate and drug delivery. A previous study has shown that intracellularly expressing CRM197EK in Escherichia coli (E. coli) host formed inclusion bodies that need a complicated purification and refolding step. Protein purification from inclusion bodies can be overcome by solubilization of inclusion bodies by using N-lauroyl sarcosine (sarkosyl). In this work, recombinant CRM197EK (rCRM197EK) was expressed in E. coli BL21 (DE3) as inclusion bodies, then solubilized using sarkosyl to form a soluble rCRM197EK without the need for a renaturation process. Furthermore, rCRM197EK was purified using the Ni-NTA column, characterized by SDS-PAGE and Western Blot, and its biological activity was assayed through its DNase activity. Moreover, its binding affinity with anti-diphtheria toxin (DT) antibody was measured using the surface plasmon resonance (SPR). The result showed that solubilization with sarkosyl form soluble rCRM197EK (61.61 kDa) was confirmed by SDS-PAGE and Western Blot with a yield of 2.8 mg/mL. rCRM197EK shows DNase activity, and the SPR assay shows that it can interact with an anti-DT antibody with a binding energy of - 9.2 kcal/mol.