[Construction and expression of vector encoding Sox2 with mutated SUMO accepter site].
OBJECTIVE
To construct eukaryotic expression plasmids encoding Sox2 and Sox2 K247R and identify their expression and SUMOylation.


METHODS
With gift plasmid 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 pCMV-Myc-SUMO1 into 293FT 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.


CONCLUSIONS
Successful construction and expression of Sox2 and Sox2 K247R. Sox2 could be SUMO lyated in vitro but Sox2 K247R not.
Guo
Ze-kun
ZK
College of Life Sciences, Northwest A & F University, Yangling 712100, China. gzkster@gmail.com
Wu
Xian-qiang
XQ
Shan
Li-ran
LR
Song
Zhen
Z
D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D055748:SOXB1 Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000378:metabolism*
D055748:SOXB1 Transcription Factors; D025842:SUMO-1 Protein