Identification of interaction site of MIC2 with aldolase in Toxoplasma gondii

VernacularTitle:刚地弓形虫MIC2与醛缩酶作用位点的鉴定
Author: Bin ZHENG ; Zhikui YIN ; Zhijun YAO ; Haizhu ZHANG ; Hongbin REN ; Ximei ZHAN
Publication Type:Journal Article
Keywords: Toxoplasma gondii; microneme protein 2 (MIC2); aldolase; site-directed mutagenesis; GST pull-down
From: Chinese Journal of Zoonoses 2014;(7):698-700,708
CountryChina
Language:Chinese
Abstract: In this study ,we aim to identify the protein interaction site of microneme protein 2 (MIC2) and aldolase in Toxoplasma gondii .The tryptophan (Trp ,W) at site 767 of carboxyl terminus of MIC2 (MIC2C) was mutated into alanine (Ala ,A) by site-directed mutagenesis to construct plasmid MIC2C W/A/pGEX-4T-1 .The mutant protein GST-MIC2C W/A was expressed in E .coli upon IPTG induction .Glutathione sepharose beads were incubated with GST-MIC2C W/A and GST-MIC2C respectively ,then incubated with tachyzoite lysates ,and bound proteins were eluted using sample buffer .Eluants were resolved by SDS-PAGE and Western blot .A protein band specifically recognized by anti-aldolase antibody was detected in prod-ucts coming from GST pull-down of GST-MIC2C ,but not in pull-down products coming from GST-MIC2C W/A .With muta-tion of MIC2C W767 to A ,MIC2 protein lost the binding ability to aldolase .Tryptophan (W767 ) was the protein interaction site of MIC2 and aldolase in T .gondii .