Parallel cloning, expression, purification and crystallization of human proteins for structural genomics

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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(TM) 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 Angstrom 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.

Detaljer

Författare
  • HT Ding
  • H Ren
  • Q Chen
  • G Fang
  • LF Li
  • R Li
  • R Wang
  • XY Jia
  • YH Liang
  • MH Hu
  • Y Li
  • JC Luo
  • XC Gu
  • Xiao-Dong Su
  • M Luo
  • SY Lu
Enheter & grupper
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Strukturbiologi
Originalspråkengelska
Sidor (från-till)2102-2108
TidskriftActa Crystallographica. Section D: Biological Crystallography
Volym58
StatusPublished - 2002
PublikationskategoriForskning
Peer review utfördJa