Abstract
Significant improvements in the technologies used for protein production have been driven by impending genome-scale proteomics projects. These initiatives have favored Escherichia coli-based expression systems, which allow rapid cloning and expression of proteins at low cost. The range of commercially available molecular biology kits, vectors, affinity tags, and host cell lines have increased dramatically in recent years. For the structural biology community, where protein production is often a rate-limiting step, these developments have made the process of producing and purifying large amounts of protein for structural studies simpler and faster. The large-scale automated screening approaches for optimizing protein production employed by structural genomics initiatives can be adapted to a more practical targeted approach appropriate for individual structural biology groups. This chapter describes simple, rapid screening methods for testing optimal vector/host combinations using a 96-well format.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Edwards, A. M., Arrowsmith, C. H., Christendat, D., Dharamsi, A., Friesen, J. D., Greenblatt, J. F., et al. (2000) Protein production: feeding the crystallographers and NMR spectroscopists. Nat. Struct. Biol. 7, 970–972.
Coughlin, P. E., Anderson, F. E., Oliver, E. J., Brown, J. M., Homans, S. W., Pollak, S., et al. (1999) Improved resolution and sensitivity of triple-resonance NMR methods for the structural analysis of proteins by use of backbone-labeling strategy. J. Am. Chem. Soc. 121, 11,871–11,874.
Creemers, A. F. L., Klaassen, C. H. W., Bovee-Geurts, P. H. M., Kelle, R., Kragl, U., Raap, J., et al. (1999) Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G-protein coupled receptor rhodopsin. Biochemistry 38, 7195–7199.
Jermutus, L., Ryabova, L. A., and Pluckthun, A. (1998) Recent advances in producing and selecting functional proteins by using cell-free translation. Curr. Opin. Biotechnol. 9, 534–548.
Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T., et al. (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 442, 15–19.
Guignard, L., Ozawa, K., Pursglove, S. E., Otting, G., and Dixon, N. E. (2002) NMR analysis of in vitro-synthesized proteins without purification: a high throughput approach. FEBS Lett. 524, 159–162.
Harwood, A. J. (1996) Basic DNA and RNA protocols. In Methods in Molecular Biology, vol. 58 (Walker, J. M., ed.). Humana, Totowa, NJ, pp.
Strausberg, R. L., Feingold, E. A., Klausner, R. D., and Collins, F. S. (1999) The mammalian gene collection. Science 286, 455–457.
Stewart, L., Clark, R., and Behnke, C. (2002) High-throughput crystallisation and structure determination in drug discovery. Drug Discov. Today 7, 187–196.
Liu, Q., Li, M. Z., Leibham, D., Cortez, D., and Elledge, S. J. (1998) The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr. Biol. 8, 1300–1309.
Studier, F. W. (1991) Use of T7 RNA polymerase to direct expression of cloned genes. Meth. Enzymol. 185, 286–299.
Miroux, B. and Walker, J. E. (1996) Overproduction of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298.
Sambrook, J. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Woodbury, NY.
Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K. (1979) Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of a single base pair mismatch. Nucleic Acids Res. 6, 3543–3557.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680–685.
Burgess, R. R. (1991) Use of polyethylenimine in purification of DNA-binding proteins. Meth. Enzymol. 208, 3–11.
Blackwell, J. R. and Horgan, R. (1991) A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Lett. 295, 10–12.
Georgiou, G. and Valax, P. (1996) Expression of correctly folded proteins in Escherichia coli. Curr. Opin. Biotechnol. 7, 190–197.
Battistoni, A., Mazzetti, A. P., and Rotilio, G. (1999) In vivo formation of Cu, Zn superoxide dismutase disulfide bond in Escherichia coli. FEBS Lett. 443, 313–316.
Davis, G. D., Elisee, C., Newham, D. M., and Harrison, R. G. (1999) New fusion protein systems designed to give soluble expression in Escherichia coli. Biotechnol. Bioeng. 65, 382–388.
Marston, F. A. O. and Hartley, D. L. (1990) Solubilization of protein aggregates. Meth. Enzymol. 182, 264–276.
Lindwall, G., Chau, M., Gardner, S. R., and Kohlstaedt, L. A. (2000) A sparse matrix approach to the solubilization of overexpressed proteins. Protein Eng. 13, 67–71.
Walker, J. M. (1996) The Protein Protocols Handbook, 1st. ed. Humana, Totowa, NJ.
Ausubel, F., Brent, R., E., K. R., Moore, a. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1999) Short protocols in molecular biology. Current Protocols in Molecular Biology. 4th ed. Wiley, Hoboken, NJ.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Hewitt, L., McDonnell, J.M. (2004). Screening and Optimizing Protein Production in E. coli . In: Downing, A.K. (eds) Protein NMR Techniques. Methods in Molecular Biology™, vol 278. Humana Press. https://doi.org/10.1385/1-59259-809-9:001
Download citation
DOI: https://doi.org/10.1385/1-59259-809-9:001
Publisher Name: Humana Press
Print ISBN: 978-1-58829-246-9
Online ISBN: 978-1-59259-809-0
eBook Packages: Springer Protocols