title: Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success.
abstract: The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure.
authors: Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA.
authors: Ryan
authors: R
authors: Choi
authors: Angela
authors: A
authors: Kelley
authors: David
authors: D
authors: Leibly
authors: Stephen Nakazawa
authors: SN
authors: Hewitt
authors: Alberto
authors: A
authors: Napuli
authors: Wesley
authors: W
authors: Van Voorhis
mesh_terms: D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D016415:Sequence Alignment
chemical_list: D011506:Proteins; D009532:Nickel