Soluble expression, purification, and stabilization of a pro-apoptotic human protein, CARP.
CARP is a novel pro-apoptotic protein that has been cloned and characterized in our previous report. Previous studies showed that suppression of CARP expression results in cell proliferation in several mammalian cell lines and over-expression of CARP leads to apoptosis and inhibition of proliferation in seven tumor cell lines [Liu et al., CARP is a novel caspase recruitment domain containing pro-apoptotic protein, Biochem. Biophys. Res. Commun. 293 (2002) 1396]. To obtain soluble and active form of CARP protein for further functional and structural studies, we have expressed CARP in Escherichia coli by using Gateway cloning system. Optimal induction and expression conditions were also studied. Recombinant histidine-tagged CARP was expressed in E. coli when the carp gene was subcloned into a Gateway expression vector pET21-DEST. The partially soluble recombinant CARP protein was purified to near homogeneity by a two-step FPLC procedure, first by Ni2+ affinity chromatography followed by a gel-filtration chromatography, which yielded about 10 mg protein/L culture with at least 95% purity. Two peaks were detected in the analytical gel-filtration chromatograph while only one peak corresponding to monomer of the CARP protein was left after adding 2 mM dithiothreitol (DTT). The polymers observed are likely due to the formation of intermolecular disulfide bridges. These results suggest that adding DTT is a good solution to prevent the formation of disulfide bonds and to stabilize the protein. Successfully growing crystals of the purified CARP protein also proved that we can produce well folded CARP protein in E. coli.
Chen
Qiang
Q
Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing 100871, China.
Hui
Rutai
R
Sun
Changhong
C
Gu
Xiaocheng
X
Luo
Ming
M
Zheng
Xiaofeng
X
D017209:Apoptosis / Q000502:physiology*; D003001:Cloning, Molecular; D003460:Crystallization; D006801:Humans; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
C491304:ANKRD1 protein, human; D009124:Muscle Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins