Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis.
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 ;low-hanging 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.
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Division of Structural Biology, University of Oxford, Wellcome Trust Centre for Human Genetics, Oxford, England.
Berrow
N S
NS
Blagova
E
E
Boucher
I W
IW
Boyle
M P
MP
Brannigan
J A
JA
Carter
L G
LG
Dierks
T
T
Folkers
G
G
Grenha
R
R
Harlos
K
K
Kaptein
R
R
Kalliomaa
A K
AK
Levdikov
V M
VM
Meier
C
C
Milioti
N
N
Moroz
O
O
Müller
A
A
Owens
R J
RJ
Rzechorzek
N
N
Sainsbury
S
S
Stuart
D I
DI
Walter
T S
TS
Waterman
D G
DG
Wilkinson
A J
AJ
Wilson
K S
KS
Zaccai
N
N
Esnouf
Robert M
RM
Fogg
Mark J
MJ
D001408:Bacillus anthracis / Q000235:genetics*; D001409:Bacillus cereus / Q000235:genetics; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D019295:Computational Biology; D003460:Crystallization; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D009682:Magnetic Resonance Spectroscopy; D040901:Proteomics / Q000379:methods*; D012343:RNA, Transfer / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012371:Robotics; D013171:Spores, Bacterial / Q000235:genetics; D013466:Sulfurtransferases
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012343:RNA, Transfer; D013466:Sulfurtransferases; C107110:ThiI protein, bacteria