High-throughput production of human proteins for crystallization: The SGC experience

Abstract

Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of ∼40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9–20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

“…Protein Expression and Purification-CNB-A (residues 137-277) and CNB-B (residues 269 -418) were cloned into pNic28-Bsa4 using ligation-independent cloning and transformed into BL21 (DE3) Escherichia coli cells (19). CNB domain mutants were cloned into the pQTEV plasmid and transformed into BL21 (DE3) E. coli cells (20).…”

Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase

“…Exonuclease I-dependent Restriction-free (ERF) Cloning of Target Genes-Target genes were cloned from genomic B. anthracis Sterne 7700 (pXO1 (34). Primers were designed based on the sequence of B. anthracis Ames, because this strain was the first to be whole genome sequenced (30).…”

“…Our major improvements of the cloning procedure included substitution of the gel purification of the PCR products in favor of treatment with exonuclease I for removal of excess primers. Furthermore, DpnI digestion was omitted because the SacB fragment present in pNIC28-BSA4 allows for negative selection against parental plasmid on plates containing 5% sucrose (34).…”

Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I

“…15,16 Protein purification is detailed in the Online Supplementary Appendix. Partial trypsin and chymotrypsin digestion was performed over concentrations from 0.8-100 mg/mL from 10 min to 3 h. Reaction mixes were analyzed by mass spectrometry and SDS-PAGE.…”