title: Salvaging Pyrococcus furiosus protein targets at SECSG.
abstract: 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.
authors.affiliation: Southeast Collaboratory for Structural Genomics, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Liu
authors.forename: Ashit K
authors.initials: AK
authors.lastname: Shah
authors.forename: Jeff E
authors.initials: JE
authors.lastname: Habel
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Ng
authors.forename: Irina
authors.initials: I
authors.lastname: Kataeva
authors.forename: Hao
authors.initials: H
authors.lastname: Xu
authors.forename: Peter
authors.initials: P
authors.lastname: Horanyi
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Jessie
authors.initials: J
authors.lastname: Chang
authors.forename: Min
authors.initials: M
authors.lastname: Zhao
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.forename: Sue
authors.initials: S
authors.lastname: Chang
authors.forename: Wolfram
authors.initials: W
authors.lastname: Tempel
authors.forename: Lirong
authors.initials: L
authors.lastname: Chen
authors.forename: Weihong
authors.initials: W
authors.lastname: Zhou
authors.forename: Doowon
authors.initials: D
authors.lastname: Lee
authors.forename: Dawei
authors.initials: D
authors.lastname: Lin
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.forename: M Gary
authors.initials: MG
authors.lastname: Newton
authors.forename: John
authors.initials: J
authors.lastname: Rose
authors.forename: Bi-Cheng
authors.initials: BC
authors.lastname: Wang
mesh_terms: 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
chemical_list: D019843:Archaeal Proteins