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Protein Science (2006), 15:2356-2365. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications

Stefanie Reich1,5, Loretto H. Puckey1,5, Caroline L. Cheetham2,5, Richard Harris2, Ammar A.E. Ali4, Uma Bhattacharyya4, Kate Maclagan2, Keith A. Powell4, Chrisostomos Prodromou3,4, Laurence H. Pearl3,4, Paul C. Driscoll2,4 and Renos Savva1,4

1 School of Crystallography, Birkbeck College, London WC1E 7HX, United Kingdom
2 Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom
3 Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, United Kingdom
4 Domainex Ltd., London SW7 3RP, United Kingdom

(RECEIVED January 6, 2006; FINAL REVISION June 20, 2006; ACCEPTED July 25, 2006)

Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error. Combinatorial Domain Hunting (CDH) is a technology for discovering soluble, highly expressed constructs of target proteins. CDH combines unbiased, finely sampled gene-fragment libraries, with a screening protocol that provides "holistic" readout of solubility and yield for thousands of protein fragments. CDH is free of the "passenger solubilization" and out-of-frame translational start artifacts of fusion-protein systems, and hits are ready for scale-up expression. As a proof of principle, we applied CDH to p85{alpha}, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.

Keywords: protein structure/folding; structure; new methods; expression systems


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Nucleic Acids Res., May 1, 2008; 36(9): e51 - e51.
[Abstract] [Full Text] [PDF]


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