A procedure for producing an anti-AXL nanobody in E. coli

Zhongyun Lan; Lingyun Li; Yili Sun; Xiangjing Meng; Zhenzhong Shi; Mengyang Du; Hui Wang; Zengchao Sun; Qianqian Cui; Lu Wang; Tengjie Geng; Siyu Zhou; Yi'ang Wang; Fangzheng Hu; Chonggang Duan; Yong Geng; Yongheng Zhu; Yuanyuan Dai

doi:10.1016/j.pep.2023.106268

A procedure for producing an anti-AXL nanobody in E. coli

Protein Expr Purif. 2023 Jul:207:106268. doi: 10.1016/j.pep.2023.106268. Epub 2023 Apr 4.

Authors

Zhongyun Lan¹, Lingyun Li², Yili Sun³, Xiangjing Meng⁴, Zhenzhong Shi⁵, Mengyang Du⁶, Hui Wang⁶, Zengchao Sun⁶, Qianqian Cui⁵, Lu Wang⁵, Tengjie Geng⁶, Siyu Zhou², Yi'ang Wang², Fangzheng Hu⁶, Chonggang Duan⁴, Yong Geng⁷, Yongheng Zhu⁸, Yuanyuan Dai⁹

Affiliations

¹ College of Food Science and Technology, Shanghai Ocean University, Shanghai, 201306, China; Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai, 201306, China; State Key Laboratory of Drug Research, The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.
² State Key Laboratory of Drug Research, The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
³ Drug Discovery Shandong Laboratory, Bohai Rim Advanced Research Institute for Drug Discovery, Yantai, Shandong, 264117, China.
⁴ Shandong Provincial Key Laboratory of Biopharmaceuticals, Shandong Academy of Pharmaceutical Sciences, Jinan, 250101, China.
⁵ State Key Laboratory of Drug Research, The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.
⁶ College of Food Science and Technology, Shanghai Ocean University, Shanghai, 201306, China; State Key Laboratory of Drug Research, The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.
⁷ State Key Laboratory of Drug Research, The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China; University of Chinese Academy of Sciences, Beijing, 100049, China. Electronic address: gengyong@simm.ac.cn.
⁸ College of Food Science and Technology, Shanghai Ocean University, Shanghai, 201306, China; Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai, 201306, China. Electronic address: yh-zhu@shou.edu.cn.
⁹ Department of Pharmacy, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100021, China; National Cancer Center/National Clinical Research Center for Cancer/Hebei Cancer Hospital, Chinese Academy of Medical Sciences, Langfang, 065001, China. Electronic address: daiyuanyuan@cicams.ac.cn.

PMID: 37023993
DOI: 10.1016/j.pep.2023.106268

Abstract

As one of the receptors of the TAM family, AXL plays a vital role in stem cell maintenance, angiogenesis, immune escape of viruses and drug resistance against tumors. In this study, the truncated extracellular segment containing two immunoglobulin-like domains of human AXL (AXL-IG), which has been confirmed to bind growth arrest specific 6 (GAS6) by structural studies [1], was expressed in a prokaryotic expression system and then purified. Immunizing camelid with the purified AXL-IG as antigen could lead to the production of unique nanobodies composed of only variable domain of heavy chain of heavy-chain antibody (VHH), which are around 15 kD and stable. We screened out a nanobody A-LY01 specific binding to AXL-IG. We further determined the affinity of A-LY01 to AXL-IG and revealed that A-LY01 could specifically recognize full-length AXL on the surface of HEK 293T/17 cells. Our study provides appropriate support for the development of diagnostic reagents and antibody therapeutics targeting AXL.

Keywords: AXL; Expression; Nanobody; Purification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antibodies
Escherichia coli* / genetics
Humans
Immunoglobulin Heavy Chains
Neoplasms*

Substances

Antibodies
Immunoglobulin Heavy Chains