Parallel cloning, expression, purification and crystallization of human proteins for structural genomics.
54 human genes were selected as test targets for parallel cloning, expression, purification and crystallization. Proteins from these genes were selected to have a molecular weight of between 14 and 50 kDa, not to have a high percentage of hydrophobic residues (i.e. more likely to be soluble) and to have no known crystal structures and were not known to be subunits of heterocomplexes. Four proteins containing transmembrane regions were selected for comparative tests. To date, 44 expression clones have been constructed with the Gateway cloning system (Invitrogen, The Netherlands). Of these, 35 clones were expressed as recombinant proteins in Escherichia coli strain BL21 (DE3)-pLysS, of which 12 were soluble and four have been purified to homogeneity. Crystallization conditions were screened for the purified proteins in 96-well plates under oil. After further refinement with the same device or by the hanging-drop method, crystals were grown, with needle, plate and prism shapes. A 2.12 A data set was collected for protein NCC27. The results provide insights into the high-throughput target selection, cloning, expression and crystallization of human genomic proteins.
Ding
Hai-tao
HT
Laboratory of Structural Biology, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China.
Ren
Hui
H
Chen
Qiang
Q
Fang
Gang
G
Li
Lan-fen
LF
Li
Rui
R
Wang
Zhuo
Z
Jia
Xiao-yu
XY
Liang
Yu-he
YH
Hu
Mei-hao
MH
Li
Yi
Y
Luo
Jing-chu
JC
Gu
Xiao-cheng
XC
Su
Xiao-dong
XD
Luo
Ming
M
Lu
Shan-yun
SY
D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D017931:DNA Primers; D023281:Genomics; D006801:Humans; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011506:Proteins; D011994:Recombinant Proteins