Advanced

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

Ding, HT; Ren, H; Chen, Q; Fang, G; Li, LF; Li, R; Wang, R; Jia, XY; Liang, YH and Hu, MH, et al. (2002) In Acta Crystallographica. Section D: Biological Crystallography 58. p.2102-2108
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
, et al. (More)
(Less)
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Acta Crystallographica. Section D: Biological Crystallography
volume
58
pages
2102 - 2108
publisher
International Union of Crystallography
external identifiers
  • pmid:12454471
  • wos:000179468800019
  • scopus:14244271957
ISSN
1399-0047
DOI
10.1107/S0907444902016359
language
English
LU publication?
yes
id
5cc7f895-08bd-4b92-8c4f-554e42699853 (old id 322953)
date added to LUP
2007-08-16 09:48:37
date last changed
2017-06-25 04:26:01
@article{5cc7f895-08bd-4b92-8c4f-554e42699853,
  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.},
  author       = {Ding, HT and Ren, H and Chen, Q and Fang, G and Li, LF and Li, R and Wang, R and Jia, XY and Liang, YH and Hu, MH and Li, Y and Luo, JC and Gu, XC and Su, Xiao-Dong and Luo, M and Lu, SY},
  issn         = {1399-0047},
  language     = {eng},
  pages        = {2102--2108},
  publisher    = {International Union of Crystallography},
  series       = {Acta Crystallographica. Section D: Biological Crystallography},
  title        = {Parallel cloning, expression, purification and crystallization of human proteins for structural genomics},
  url          = {http://dx.doi.org/10.1107/S0907444902016359},
  volume       = {58},
  year         = {2002},
}