Expression screening, protein purification and NMR analysis of human protein domains for structural genomics.
Structural genomics, the determination of protein structures on a genome-wide scale, is still in its infancy for eukaryotes due to the number and size of their genes. Low protein expression and solubility of eukaryotic geneproducts are the major bottlenecks in high-throughput (HTP) recombinant protein production with the E. coli expression systems. To circumvent this problem we decided to focus on separate protein domains. We describe here a fast microtiterplate based, expression and solubility screening procedure, using a combination of in vitro and in vivo expression, and purification with nickel-NTA magnetic beads. All steps are optimized for automatic HTP processing using a liquid handling station. Furthermore, large-scale expression and protein purification conditions are optimized, permitting the purification of 24 protein samples per week. We further show that results obtained from the expression screening can be extrapolated to the production of protein samples for NMR. Starting with 81 cloned human protein domains, in vivo expression was detected in 54 cases, and from 28 of those milligrams of protein were purified. An informative HSQC spectrum was recorded for 18 proteins (22%), half of which were indicative of a folded protein. The success rate and quality of the HSQC spectra suggest that the domain approach holds promise for human proteins.
Folkers
G E
GE
Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. gert@nmr.chem.uu.nl
van Buuren
B N M
BN
Kaptein
R
R
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D023281:Genomics; D006801:Humans; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D040901:Proteomics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D012995:Solubility
D011994:Recombinant Proteins