Salvaging Pyrococcus furiosus protein targets at SECSG.
Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2,200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.
Liu
Zhi-Jie
ZJ
Southeast Collaboratory for Structural Genomics, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Shah
Ashit K
AK
Habel
Jeff E
JE
Ng
Joseph D
JD
Kataeva
Irina
I
Xu
Hao
H
Horanyi
Peter
P
Yang
Hua
H
Chang
Jessie
J
Zhao
Min
M
Huang
Lei
L
Chang
Sue
S
Tempel
Wolfram
W
Chen
Lirong
L
Zhou
Weihong
W
Lee
Doowon
D
Lin
Dawei
D
Zhang
Hua
H
Newton
M Gary
MG
Rose
John
J
Wang
Bi-Cheng
BC
D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003461:Crystallography / Q000379:methods; D015870:Gene Expression; D008958:Models, Molecular; D040901:Proteomics / Q000379:methods*; D020027:Pyrococcus furiosus / Q000737:chemistry*; D014961:X-Ray Diffraction / Q000379:methods
D019843:Archaeal Proteins