Development in Detection Methods for the Expression of Surface-Displayed Proteins

Frontiers in Microbiology

Ronggui Li¹  Shuhao Li²  Lei Li²  Chenglong Ma²  Dongping Zhao²  Chunyang Jiang² 

[1] College of Life Sciences, Qingdao University, Qingdao, China;School of Basic Medicine, Qingdao Medical College, Qingdao University, Qingdao, China;

关键词: ELISA;    molecular display platform;    phage display;    SH2;    chromodomain;   

DOI  :  10.3389/fmicb.2022.899578

来源: DOAJ

【 摘 要 】

Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. A protein of interest is selected as the template and expressed on a molecular display platform such as a bacteriophage for engineering. Initially, the surface-displayed protein template needs to be checked against the desired target via ELISA to examine whether the functions of the displayed template remain intact. The ELISA signal is subject to the protein-target binding affinity. A low-affinity results in a weak ELISA signal which makes it difficult to determine whether the weak signal is because of low affinity or because of poor expression of the protein. Using a methyllysine-binding chromodomain protein Cbx1 that weakly binds to the histone H3K9me3 peptide, we developed and compared three different approaches to increase the signal-to-background ratio of ELISA measurements. We observed that the specific peptide-binding signal was enhanced by increasing the Cbx1 phage concentration on the ELISA plate. The introduction of previously known gain-of-function mutations to the Cbx1 protein significantly increased the ELISA signals. Moreover, we demonstrated that the H3K9me3-specific binding signal was enhanced by fusing Cbx1 with a high-affinity phosphotyrosine-binding protein and by coating the ELISA plate with a mixture of H3K9me3 and phosphotyrosine peptides. This approach also worked with binding to a lower affinity momomethyllysine peptide H3K9me1. These approaches may help improve ELISA experiments when dealing with low-affinity ligand-protein interactions.

【 授权许可】

Unknown