[Cloning, expression, purification and identification of multi-potent transcription factor CTCF recombinant and its segments].
OBJECTIVE
To clone ctc f cDNA, N, Zn and C fragments separately into expresstion vector, purify and identify the expressed proteins.


METHODS
Using the recombinant plasmid pGEM7Zf (-)-ctc f as template for PCR, pGEX-4T-2-ctc f, pGEX-4T-2-ctc f-N, pGEX-4T-2- ctc f-Zn and pGEX-4T-2- ctc f-C recombinants were constructed successfully. After transformed into E. coli BL21 cells, the recombinants were confirmed by enzyme digestion and sequence analysis. After optimizing the IPTG inducing condition, the purified GST fusion proteins with affinity chromatography were conformed by Far-Western blotting.


RESULTS
The recombinant plasmids pGEX-4T-2-ctc f, pGEX-4T-2- ctc f-N, pGEX-4T-2-ctc f-Zn and pGEX-4T-2-ctc f-C were confirmed by restriction enzyme assay and sequencing. All GST fusion proteins, CTCF, CTCF-N, CTCF-Zn and CTCF-C were successfully expressed at the optimal parameters and purified with affinity chromatography, and specifically recognized by anti-GST antibody.


CONCLUSIONS
Ctc f, ctc f-n, ctc f-Zn and ctc f-c gene recombinants were constructed successfully and their corresponding fusion proteins were successfully purified with affinity chromatography and identified.
Jiang
Lei
L
Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Qin
Yang
Y
Sun
Zhi-Lin
ZL
Wu
Jing
J
Yang
Wen-Li
WL
Zhang
Jun-Zhe
JZ
D000076246:CCCTC-Binding Factor; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D005982:Glutathione Transferase