Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease

Yibing Deng; Shengchao Shi; Bin Zheng; Tao Wu; Peng Zheng

doi:10.21769/BioProtoc.3596

Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease

Bio Protoc. 2020 Apr 20;10(8):e3596. doi: 10.21769/BioProtoc.3596.

Authors

Yibing Deng¹, Shengchao Shi¹, Bin Zheng¹, Tao Wu¹, Peng Zheng¹

Affiliation

¹ State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, 21002, China.

Abstract

The development of chemical and biological coupling technologies in recent years has made possible of protein polymers engineering. We have developed an enzymatic method for building polyproteins using a protein ligase OaAEP1 (asparagine endopeptidase 1) and protease TEV (tobacco etching virus). Using a mobile TEV protease site compatible with the OaAEP1 ligation, we achieved a stepwise polymerization of the protein on the surface. The produced polyprotein can be verified by protein unfolding scenario using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides an alternative method for polyprotein engineering and immobilization.

Keywords: Enzyme; OaAEP1; Polymerization; Polyprotein engineering; Single-molecule force spectroscopy.