Purification and activity of anti-PD-L1&CXCR4 bispecific nanobody
10.11665/j.issn.1000-5048.20210516
- VernacularTitle:Anti-PD-L1&CXCR4双特异性纳米抗体的纯化与活性
- Author:
Shuyi XU
1
;
Yaxian LI
;
Hai HU
;
Li ZHANG
;
Yanlin BIAN
;
Jianwei ZHU
;
Mingyuan WU
Author Information
1. 上海交通大学药学院细胞工程及抗体药物教育部工程研究中心
- Publication Type:Journal Article
- Keywords:
bispecific;
nanobody;
affinity chromatography;
protein purification;
biological activity
- From:
Journal of China Pharmaceutical University
2021;52(5):622-629
- CountryChina
- Language:Chinese
-
Abstract:
Targeted programmed death-ligand 1 (PD-L1) and CXC chemokine receptor type 4 (CXCR4), gene sequences encoding anti-PD-L1 nanobody and anti-CXCR4 nanobody were cloned into the pET-22b (+) vector to construct recombinant expression plasmid of anti-PD-L1&CXCR4 bispecific nanobody, which was connected with 6 × His tag and transformed into E.coli BL21 (DE3). The expressed proteins were then found to exist as a soluble form in the supernatant of bacterial lysate after induction of IPTG.Three purification methods were used to obtain the target protein in order to improve the yield and purity of the bispecific nanobody.The bacterial supernatant was separated and purified by His Trap FF affinity chromatographic column.The target protein output could exceed 1 mg/L, and the product purity could reach up to 97%.Besides, the anti-PD-L1&CXCR4 bispecific nanobody shows a specific binding ability to two antigens on the cell surface, enhancing the cytotoxicity of IL-2 activated human peripheral blood mononuclear cells (PBMC) to tumor cell line AsPC-1, which lays the foundation for further evaluation of its drug efficacy in vivo.
