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  • Macro-to-micro structural proteomics: native source proteins for high-throughput crystallization.

Macro-to-micro structural proteomics: native source proteins for high-throughput crystallization.

PloS one (2012-03-07)
Monica Totir, Nathaniel Echols, Max Nanao, Christine L Gee, Alisa Moskaleva, Scott Gradia, Anthony T Iavarone, James M Berger, Andrew P May, Chloe Zubieta, Tom Alber
ABSTRACT

Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Phosphoglucose Isomerase from Bacillus stearothermophilus, lyophilized powder, 300-1,000 units/mg protein
Supelco
Phosphoglucose Isomerase from baker′s yeast (S. cerevisiae), for use with Fructose Assay Kit FA-20
Sigma-Aldrich
Phosphoglucose Isomerase from baker′s yeast (S. cerevisiae), Type III, ammonium sulfate suspension, ≥400 units/mg protein (biuret)
Sigma-Aldrich
Phosphoglucose Isomerase from rabbit muscle, Type XI, lyophilized powder, ≥200 units/mg protein