Expression, purification and characterization of anti-α2δ1/NanoLuc fusion protein.

Lina MA; Qi HE; Jiangli XU; Zhiqian Zhang

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Expression, purification and characterization of anti-α2δ1/NanoLuc fusion protein.

Author: Lina MA ¹ ; Qi HE ¹ ; Jiangli XU ² ; Zhiqian ZHANG ¹
Author Information

1. Key Laboratory of Clinical Laboratory Diagnostics of Ministry Education, Faculty of Laboratory Medicine, Chongqing Medicine University, Chongqing 400016, China.
2. Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Cell Biology, Peking University Cancer Hospital and Institute, Beijing 100142, China.
Publication Type:Journal Article
Keywords: NanoLuc; fusion protein; monoclonal antibody; α2δ1
MeSH: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Neoplastic Stem Cells; Recombinant Proteins/genetics*; Single-Chain Antibodies/genetics*
From: Chinese Journal of Biotechnology 2021;37(11):4124-4133
CountryChina
Language:Chinese
Abstract: The existence of cancer stem cells is regarded as the major cause for therapeutic resistance and relapse of a variety of cancer types including hepatocellular carcinoma (HCC). However, the tracing of such a subpopulation in vivo has been challenging. We have previously demonstrated that the isoform 5 of the voltage-gated calcium channel α2δ1 subunit, which can be recognized specifically by a monoclonal antibody 1B50-1, is a bona fide surface marker for HCC stem cells. Here we developed a strategy for optical imaging of α2δ1-positive cells by using a fusion protein containing the single chain variable fragment (scFv) of Mab1B50-1 and the luciferase NanoLuc which was tagged with Flag in the C-terminal. The scFv of Mab1B50-1 was fused to the N-terminal of NanoLucFlag using overlap PCR, and the recombinant fragment, which was named as 1B50-1scFv-NanoLucFlag, was subsequently cloned into a eukaryotic expression vector. The resulting construct was transfected into FreeStyle 293F cells in suspension using PEI reagent. The expression of the fusion protein was identified as a protein with molecular weight about 50 kDa by Western blotting. After purification by ANTI-FLAG® M2 affinity chromatography, 1B50-1scFv-NanoLucFlag was demonstrated to bind to α2δ1 positive cells specifically with a Kd value of (18.62±1.84) nmol/L. Furthermore, a strong luciferase activity of 1B50-1scFv-NanoLucFlag was detected in α2δ1 positive cells following incubation with the fusion protein, indicating that the presence of α2δ1 could be quantified using this fusion protein. Hence, 1B50-1scFv-NanoLucFlag provides a potential tool for optical imaging of α2δ1 positive cancer stem cells both in vitro and in vivo.