Purification of a bioactive recombinant human Reg IV expressed in Escherichia coli.
Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily. Reg IV is highly expressed in the gastrointestinal tract and markedly up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel disease. However, the physiological and pathological functions of Reg IV are largely unknown, partly due to the limited access of the bioactive protein. We report here the first expression and purification of Reg IV proteins using a prokaryotic system. Human Reg IV was expressed in Escherichia coli as an insoluble protein which was identified in the fraction of inclusion body after ultrasonication of the bacteria. After the protein aggregate was solubilized by guanidine-HCl, it was refolded by sucrose and arginine-assisted procedures and purified using cation-exchange chromatography. The protein identity and purity of the final preparation were confirmed by analysis of the protein mass and immune specificity in SDS-PAGE, Western blotting, and HPLC assay. The biological activity of the protein was determined by the HCT116 and HT29 cell proliferation assays. The highly purified bioactive human Reg IV should aid in further characterization of its physiological and pathological functions.
Hu
Guoyong
G
Department of Gastroenterology, Shanghai First People's Hospital, Institute of Pancreas Diseases, Shanghai Jiao Tong University, Shanghai 200080, PR China.
Shen
Jiaqin
J
Cheng
Li
L
Xiang
Di
D
Zhang
Zhonghui
Z
He
Miao
M
Lu
Huili
H
Zhu
Shunying
S
Wu
Mingyuan
M
Yu
Yan
Y
Wang
Xingpeng
X
Han
Wei
W
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D049109:Cell Proliferation; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D037181:Lectins, C-Type / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; C428528:REG4 protein, human; D011994:Recombinant Proteins