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

K. Au; N. S. Berrow; E. Blagova; I. W. Boucher; M. P. Boyle; J. A. Brannigan; L. G. Carter; T. Dierks; G. Folkers; R. Grenha; K. Harlos; R. Kaptein; A. K. Kalliomaa; V. M. Levdikov; C. Meier; N. Milioti; O. Moroz; A. Muller; R. J. Owens; N. Rzechorzek; S. Sainsbury; D. I. Stuart; T. S. Walter; D. G. Waterman; A. J. Wilkinson; K. S. Wilson; N. Zaccai; Robert M. Esnouf; Mark J. Fogg

doi:10.1107/S0907444906033555

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

K. Au, N. S. Berrow, E. Blagova, I. W. Boucher, M. P. Boyle, J. A. Brannigan, L. G. Carter, T. Dierks, G. Folkers, R. Grenha, K. Harlos, R. Kaptein, A. K. Kalliomaa, V. M. Levdikov, C. Meier, N. Milioti, O. Moroz, A. Muller, R. J. Owens, N. RzechorzekS. Sainsbury, D. I. Stuart, T. S. Walter, D. G. Waterman, A. J. Wilkinson, K. S. Wilson, N. Zaccai, Robert M. Esnouf, Mark J. Fogg

Chemistry

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Pages (from-to)	1267-1275
Number of pages	9
Journal	Acta Crystallographica. Section D, Biological Crystallography
Volume	62
DOIs	https://doi.org/10.1107/S0907444906033555
Publication status	Published - Oct 2006

Keywords

NANOLITRE CRYSTALLIZATION EXPERIMENTS
GENOME SEQUENCE
D-RIBULOSE-5-PHOSPHATE 3-EPIMERASE
PROTEIN
MECHANISM
SUBTILIS
SPORULATION
CEREUS
DOMAIN
SPO0A

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10.1107/S0907444906033555

Cite this

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., ... Fogg, M. J. (2006). Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis. Acta Crystallographica. Section D, Biological Crystallography, 62, 1267-1275. https://doi.org/10.1107/S0907444906033555

@article{79f7783c6ecd41bf87ac3057af47fea9,

title = "Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis",

abstract = "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.",

keywords = "NANOLITRE CRYSTALLIZATION EXPERIMENTS, GENOME SEQUENCE, D-RIBULOSE-5-PHOSPHATE 3-EPIMERASE, PROTEIN, MECHANISM, SUBTILIS, SPORULATION, CEREUS, DOMAIN, SPO0A",

author = "K. Au and Berrow, {N. S.} and E. Blagova and Boucher, {I. W.} and Boyle, {M. P.} and Brannigan, {J. A.} and Carter, {L. G.} and T. Dierks and G. Folkers and R. Grenha and K. Harlos and R. Kaptein and Kalliomaa, {A. K.} and Levdikov, {V. M.} and C. Meier and N. Milioti and O. Moroz and A. Muller and Owens, {R. J.} and N. Rzechorzek and S. Sainsbury and Stuart, {D. I.} and Walter, {T. S.} and Waterman, {D. G.} and Wilkinson, {A. J.} and Wilson, {K. S.} and N. Zaccai and Esnouf, {Robert M.} and Fogg, {Mark J.}",

year = "2006",

month = oct,

doi = "10.1107/S0907444906033555",

language = "English",

volume = "62",

pages = "1267--1275",

journal = "Acta Crystallographica. Section D, Biological Crystallography",

issn = "0907-4449",

publisher = "International Union of Crystallography",

}

Au, K, Berrow, NS, Blagova, E, Boucher, IW, Boyle, MP, Brannigan, JA, Carter, LG, Dierks, T, Folkers, G, Grenha, R, Harlos, K, Kaptein, R, Kalliomaa, AK, Levdikov, VM, Meier, C, Milioti, N, Moroz, O, Muller, A, Owens, RJ, Rzechorzek, N, Sainsbury, S, Stuart, DI, Walter, TS, Waterman, DG, Wilkinson, AJ , Wilson, KS, Zaccai, N, Esnouf, RM & Fogg, MJ 2006, 'Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis', Acta Crystallographica. Section D, Biological Crystallography, vol. 62, pp. 1267-1275. https://doi.org/10.1107/S0907444906033555

TY - JOUR

T1 - Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis

AU - Au, K.

AU - Berrow, N. S.

AU - Blagova, E.

AU - Boucher, I. W.

AU - Boyle, M. P.

AU - Brannigan, J. A.

AU - Carter, L. G.

AU - Dierks, T.

AU - Folkers, G.

AU - Grenha, R.

AU - Harlos, K.

AU - Kaptein, R.

AU - Kalliomaa, A. K.

AU - Levdikov, V. M.

AU - Meier, C.

AU - Milioti, N.

AU - Moroz, O.

AU - Muller, A.

AU - Owens, R. J.

AU - Rzechorzek, N.

AU - Sainsbury, S.

AU - Stuart, D. I.

AU - Walter, T. S.

AU - Waterman, D. G.

AU - Wilkinson, A. J.

AU - Wilson, K. S.

AU - Zaccai, N.

AU - Esnouf, Robert M.

AU - Fogg, Mark J.

PY - 2006/10

Y1 - 2006/10

N2 - 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.

AB - 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.

KW - NANOLITRE CRYSTALLIZATION EXPERIMENTS

KW - GENOME SEQUENCE

KW - D-RIBULOSE-5-PHOSPHATE 3-EPIMERASE

KW - PROTEIN

KW - MECHANISM

KW - SUBTILIS

KW - SPORULATION

KW - CEREUS

KW - DOMAIN

KW - SPO0A

U2 - 10.1107/S0907444906033555

DO - 10.1107/S0907444906033555

M3 - Article

SN - 0907-4449

VL - 62

SP - 1267

EP - 1275

JO - Acta Crystallographica. Section D, Biological Crystallography

JF - Acta Crystallographica. Section D, Biological Crystallography

ER -