Construction and expression of vector encoding Sox2 with mutated SUMO accepter site

VernacularTitle:转录因子Sox2 SUMO修饰位点突变体的构建及真核表达
Author: Zekun GUO ; Xianqiang WU ; Liran SHAN ; Zhen SONG
Publication Type:Journal Article
Keywords: Sox2; SUMO; subcloning; site-directed mutagenesis; eukaryotic expression
From: Journal of Cellular and Molecular Immunology 2009;25(11):967-969
CountryChina
Language:Chinese
Abstract: AIM:To construct eukaryotic expression plasmids encoding Sox2 and Sox2 K247R and identify their expression and SUMOylation. METHODS: With gift plas-mid encoding Sox2 gene as a template, Sox2 K247R was obtained by overlapping extension PCR, followed by construction of pCMV-HA-Sox2 and pCMV-HA-Sox2 K247R. After enzyme digestion analysis and DNA sequencing, these two constructs were transfected or co-transfected with pC-MV-Myc-SUMO1 into 293 FT cells by lipofectin method. Western blot was employed to analyze expression and SUMO ylation of Sox2. RESULTS: It was revealed that eukaryotic expression vectors were constructed with correct sequence, where in mutant Sox2, the AAG codon was switched to CGG codon. Western blot results showed that good expression of both wt and mut Sox2, of which the latter could not be modified by SUMO1. CONCLUSION: Successful construction and expression of Sox2 and Sox2 K247R. Sox2 could be SUMO lyated in vitro but Sox2 K247R not.