Soluble expression of human DRR1 (down-regulated in renal cell carcinoma 1) in Escherichia coli and preparation of its polyclonal antibodies.
Human DRR1 (down-regulated in renal cell carcinoma 1) is widely expressed in normal tissues but dramatically reduced or even undetectable in a number of different cancer cell lines and primary tumour types. DRR1 from Homo sapiens was cloned into the pQE30 vector for fusion-protein expression with six histidine residues in Escherichia coli BL21(DE3). A soluble protein with a molecular mass of approx. 19 kDa on SDS/PAGE that matches the expected rDRR1 (recombinant DRR1) molecular mass (18.7 kDa) was obtained. The soluble and insoluble expression of recombinant protein DRR1 (rDRR1) was temperature-dependent. The expression rDRR1 was in soluble and insoluble forms at 37 degrees C, and approx. 80% of total rDRR1 was soluble at 37 degrees C, while rDRR1 was almost exclusively expressing in soluble form at 20 degrees C. The expressed rDRR1 at 20 degrees C was affinity-purified on Ni(2+)-charged resin under native conditions. The purified protein was further identified by ESI-MS (electrospray ionization MS). The purified recombinant protein rDRR1 was further used to raise anti-(human DRR1) polyclonal antibodies, which were suitable for detecting both the recombinant exogenous DRR1 and the endogenous DRR1 from tissues and cells by immunoblotting and immunohistochemistry. The purified rDRR1 and our prepared anti-(human DRR1) polyclonal antibodies may provide useful tools for future biological function studies on DRR1.
Zhao
Xin-Yu
XY
State Key Laboratory of Biotherapy, West China Hospital and School of Life Sciences, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
Li
Hong-Xia
HX
Liang
Shu-Fang
SF
Yuan
Zhu
Z
Yan
Fei
F
Ruan
Xu-Zhi
XZ
You
Jia
J
Xiong
Shao-Quan
SQ
Tang
Ming-Hai
MH
Wei
Yu-Quan
YQ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016466:CHO Cells; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli; D016147:Genes, Tumor Suppressor; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization
D000906:Antibodies; C403503:FAM107A protein, human; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins