Prokaryotic expression, purification and functional characterization of human FHL3.
Four and a half LIM domain protein 3 (FHL3) is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. The full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression vector pBV220 and then the recombinant plasmid was transformed into E. coli JM109. The expression of the recombinant protein was induced at 42 degrees C. SDS-PAGE analysis showed that recombinant human FHL3 (rhFHL3) was mainly expressed as an inclusion body. After purification by HisTrap FF crude, the rhFHL3 was renatured by dialysis against renaturing buffer and identified by Western blot analysis using human FHL3 polyclonal antibody. The MTT assay showed that the purified rhFHL3 could inhibit HepG2 cell growth but promote the proliferation of ECV304 cells. In addition, the expression of angiogenin (Ang) gene was increased when ECV304 cells were pretreated with rhFHL3.
Huang
Xin
X
Laboratory of Molecular Genetics, Beijing Institute of Basic Medical Sciences, Beijing, 100850, China.
Wang
Jinfeng
J
Xia
Wenrong
W
Zou
Minji
M
Xu
Tao
T
Jin
Zhe
Z
Cai
Xin
X
Wang
Yuanyuan
Y
Wang
Jiaxi
J
Xu
Donggang
D
D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D022781:Hepatocytes / Q000187:drug effects; D006801:Humans; D002479:Inclusion Bodies; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D060588:LIM Domain Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013154:Spleen; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
D018076:DNA, Complementary; C117563:FHL3 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue