title: Parallel cloning, expression, purification and crystallization of human proteins for structural genomics.
abstract: 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.
authors.affiliation: Laboratory of Structural Biology, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China.
authors.forename: Hai-tao
authors.initials: HT
authors.lastname: Ding
authors.forename: Hui
authors.initials: H
authors.lastname: Ren
authors.forename: Qiang
authors.initials: Q
authors.lastname: Chen
authors.forename: Gang
authors.initials: G
authors.lastname: Fang
authors.forename: Lan-fen
authors.initials: LF
authors.lastname: Li
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Wang
authors.forename: Xiao-yu
authors.initials: XY
authors.lastname: Jia
authors.forename: Yu-he
authors.initials: YH
authors.lastname: Liang
authors.forename: Mei-hao
authors.initials: MH
authors.lastname: Hu
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.forename: Jing-chu
authors.initials: JC
authors.lastname: Luo
authors.forename: Xiao-cheng
authors.initials: XC
authors.lastname: Gu
authors.forename: Xiao-dong
authors.initials: XD
authors.lastname: Su
authors.forename: Ming
authors.initials: M
authors.lastname: Luo
authors.forename: Shan-yun
authors.initials: SY
authors.lastname: Lu
mesh_terms: 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
chemical_list: D017931:DNA Primers; D011506:Proteins; D011994:Recombinant Proteins