[Prokaryotic expression and purification of N-terminal and C-terminal fragments of histone deacetylase 4].
OBJECTIVE
To express and purify the fusion proteins of glutathione S-transferase (GST)-N-terminal of histone deacetylase4 (HDAC4-N') (1-1952 bp) and GST- C-terminal of HDAC4 (HDAC4-C') (1708-3255 bp) in E.coli.


METHODS
The DNA fragments (HDAC4-N' and HDAC4-C') amplified by PCR were ligated into GST fusion vector (pGEX-6P-1) to construct the recombinant plasmids. After identification with restriction digestion and DNA sequencing, the recombinant plasmids were transformed into E.coli BL21 and induced by IPTG for their expression. After identification by SDS-PAGE and Western blotting, the target proteins were purified by glutathione sepharose 4B.


RESULTS
The results of restriction digestion and DNA sequencing confirmed successful construction of the recombinant plasmids. The relative molecular masses of the fusion proteins were approximately 110500 and 93080 as shown by SDS-PAGE. Western blotting demonstrated that the fusion proteins could be recognized by the specific anti-HDAC4 antibody.


CONCLUSIONS
We have successfully constructed the recombinant expression vectors of pGEX-6P-1/HDAC4-N' and pGEX-6P-1/HDAC4-C' and induced the expression of the fusion proteins, which may facilitate functional studies of HDAC4 with other proteins.
Yang
Yang
Y
Department of Neurobiology, Southern Medical University, Guangzhou 510515, China. yangyang_story@126.com
Qin
Xiao-cui
XC
Liu
Shu-hu
SH
Huang
Wei
W
Wang
Xue-min
XM
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006655:Histone Deacetylases / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D005982:Glutathione Transferase; C400300:HDAC4 protein, human; D006655:Histone Deacetylases