[Expression and purification of rat betacellulin with biological activity].
OBJECTIVE
To obtain recombinant rat betacellulin with biological activity.


METHODS
A 534 bp of rat betacellulin gene fragment was amplified by RT-PCR from rat kidney and cloned into pET28a(+) vector to construct recombinant plasmid pET28a-rBTC. The recombinant plasmid was transformed into E. coli BL-21(DE3) and the betacellulin was expressed under IPTG induction. The expressed betacellulin was detected by SDS-PAGE and Western blot. The expressed protein was purified by Ni(2+) affinity chromatography and then renatured by dialysis. The effect of the renatured protein on proliferation of NIH3T3 cells was detected by MTT colorimetry.


RESULTS
Rat betacellulin protein with M(r) being 20 000 was expressed under IPTG induction. The purity of purified protein reached over 96%. After renaturation, the expressed protein could significantly stimulate the proliferation of NIH3T3 cells.


CONCLUSIONS
Rat betacellulin gene is successfully cloned into the expression vector pET28a(+) and highly expressed in E.coli. Purified and refolded betacellulin protein can obviously stimulate the proliferation of NIH3T3 cells.
An
Jia-ze
JZ
Department of Hepato-Biliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. anchen@fmmu.edu.cn
Song
Zhen-shun
ZS
Dou
Ke-feng
KF
Li
Kai-zong
KZ
Han
Hua
H
D000818:Animals; D066259:Betacellulin; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D016133:Polymerase Chain Reaction; D020673:Protein Renaturation; D051381:Rats; D017422:Sequence Analysis, DNA
D066259:Betacellulin; C587852:Btc protein, mouse; C587853:Btc protein, rat; D036341:Intercellular Signaling Peptides and Proteins