Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis

被引：23

作者：

Au, K.

Berrow, N. S.

Blagova, E.

Boucher, I. W.

Boyle, M. P.

Brannigan, J. A.

Carter, L. G.

Dierks, T.

Folkers, G.

Grenha, R.

Harlos, K.

Kaptein, R.

Kalliomaa, A. K.

Levdikov, V. M.

Meier, C.

Milioti, N.

Moroz, O.

Muller, A.

Owens, R. J.

Rzechorzek, N.

Sainsbury, S.

Stuart, D. I.

Walter, T. S.

Waterman, D. G.

Wilkinson, A. J.

Wilson, K. S.

Zaccai, N.

Esnouf, Robert M.

Fogg, Mark J.

机构：

[1] Univ Oxford, Div Struct Biol, Wellcome Trust Ctr Human Genet, Oxford OX3 7BN, England

[2] Univ York, York Struct Biol Lab, Dept Chem, York YO10 5YW, N Yorkshire, England

[3] Univ Utrecht, Bijvoet Ctr Biomol Res, NMR Spect, NL-3584 CH Utrecht, Netherlands

来源：

ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY | 2006年 / 62卷

基金：

英国医学研究理事会;

关键词：

D O I：

10.1107/S0907444906033555

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A collaborative project between two Structural Proteomics In Europe ( SPINE) partner laboratories, York and Oxford, aimed at high- throughput ( HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target- selection strategy combining ` lowhanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation- independent cloning ( LIC) and expression systems, employing custom LIC- PCR, Gateway and In- Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X- ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target- selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

引用

页码：1267 / 1275

页数：9

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