Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004;5(1-2):119-31.
doi: 10.1023/B:JSFG.0000029200.66197.0c.

Expression screening, protein purification and NMR analysis of human protein domains for structural genomics

G E Folkers  1 , B N M van Buuren, R Kaptein
Affiliations

Expression screening, protein purification and NMR analysis of human protein domains for structural genomics

G E Folkers et al. J Struct Funct Genomics. 2004.

Abstract

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.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2654-9 - PubMed
    1. Biochemistry. 2002 Jan 8;41(1):1-7 - PubMed
    1. Nat Struct Biol. 2000 Nov;7 Suppl:970-2 - PubMed
    1. Curr Opin Chem Biol. 2003 Feb;7(1):39-43 - PubMed
    1. Nature. 2003 Mar 13;422(6928):216-25 - PubMed

Publication types

Substances

LinkOut - more resources