Fast Identification of Folded Human Protein Domains Expressed in E. Coli Suitable for Structural Analysis

Overview

Journal BMC Struct Biol

Publisher Biomed Central

Specialties Biochemistry
Molecular Biology

Date 2004 Apr 29

PMID 15113422

Citations 9

Authors

Christoph Scheich

Dietmar Leitner

Volker Sievert

Martina Leidert

Brigitte Schlegel

Bernd Simon

Ivica Letunic

Konrad Bussow

Anne Diehl

Affiliations

Soon will be listed here.

Abstract

Background: High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination.

Results: 88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC) was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains) were quickly identified as being well folded and suitable for structural analysis.

Conclusion: The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.

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References

Schultz J, Copley R, Doerks T, Ponting C, Bork P . SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res. 1999; 28(1):231-4. PMC: 102444. DOI: 10.1093/nar/28.1.231. View

Bussow K, Cahill D, Nietfeld W, Bancroft D, Scherzinger E, Lehrach H . A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library. Nucleic Acids Res. 1998; 26(21):5007-8. PMC: 147919. DOI: 10.1093/nar/26.21.5007. View

Letunic I, Goodstadt L, Dickens N, Doerks T, Schultz J, Mott R . Recent improvements to the SMART domain-based sequence annotation resource. Nucleic Acids Res. 2001; 30(1):242-4. PMC: 99073. DOI: 10.1093/nar/30.1.242. View

Buchanan S, Sauder J, Harris T . The promise of structural genomics in the discovery of new antimicrobial agents. Curr Pharm Des. 2002; 8(13):1173-88. DOI: 10.2174/1381612023394809. View

Rehm T, Huber R, Holak T . Application of NMR in structural proteomics: screening for proteins amenable to structural analysis. Structure. 2002; 10(12):1613-8. DOI: 10.1016/s0969-2126(02)00894-8. View

Zhang C, Kim S . Overview of structural genomics: from structure to function. Curr Opin Chem Biol. 2003; 7(1):28-32. DOI: 10.1016/s1367-5931(02)00015-7. View

Kim M, Cierpicki T, Derewenda U, Krowarsch D, Feng Y, Devedjiev Y . The DCX-domain tandems of doublecortin and doublecortin-like kinase. Nat Struct Biol. 2003; 10(5):324-33. DOI: 10.1038/nsb918. View

Woestenenk E, Hammarstrom M, Hard T, Berglund H . Screening methods to determine biophysical properties of proteins in structural genomics. Anal Biochem. 2003; 318(1):71-9. DOI: 10.1016/s0003-2697(03)00162-3. View

Scheich C, Sievert V, Bussow K . An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography. BMC Biotechnol. 2003; 3:12. PMC: 183854. DOI: 10.1186/1472-6750-3-12. View

10.

Scheich C, Niesen F, Seckler R, Bussow K . An automated in vitro protein folding screen applied to a human dynactin subunit. Protein Sci. 2004; 13(2):370-80. PMC: 2286713. DOI: 10.1110/ps.03304604. View

11.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

12.

Inoue H, Nojima H, Okayama H . High efficiency transformation of Escherichia coli with plasmids. Gene. 1990; 96(1):23-8. DOI: 10.1016/0378-1119(90)90336-p. View

13.

Piotto M, Saudek V, SKLENAR V . Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR. 1992; 2(6):661-5. DOI: 10.1007/BF02192855. View

14.

Okajima T, Tanabe T, Yasuda T . Nonurea sodium dodecyl sulfate-polyacrylamide gel electrophoresis with high-molarity buffers for the separation of proteins and peptides. Anal Biochem. 1993; 211(2):293-300. DOI: 10.1006/abio.1993.1272. View

15.

Gronenborn A, Clore G . Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy. Protein Sci. 1996; 5(1):174-7. PMC: 2143249. DOI: 10.1002/pro.5560050123. View

16.

Bussow K, Nordhoff E, Lubbert C, Lehrach H, Walter G . A human cDNA library for high-throughput protein expression screening. Genomics. 2000; 65(1):1-8. DOI: 10.1006/geno.2000.6141. View