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Reich, Stephanie, Puckey, Loretto H, Cheetham, Caroline L, Harris, Richard, Ali, Ammar A E, Bhattacharyya, Uma, Maclagan, Kate, Powell, Keith A, Prodromou, Chrisostomos, Pearl, Laurence H, Driscoll, Paul C and Savva, Renos (2006) Combinatorial domain hunting: an effective approach for the identification of soluble protein domains adaptable to high-throughput applications. Protein Science, 15 (10). pp. 2356-2365. ISSN 0961-8368
Full text not available from this repository.Abstract
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 p85alpha, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.
| Item Type: | Article |
|---|---|
| Keywords: | *Combinatorial Chemistry Techniques Gene Library Peptide Fragments/chemistry/isolation & purification *Protein Structure, Tertiary Proteins/*chemistry/isolation & purification Solubility |
| Schools and Departments: | School of Life Sciences > Biochemistry |
| Subjects: | Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry T Technology > TP Chemical technology > TP0248.13 Biotechnology |
| Depositing User: | Chrisostomos Prodromou |
| Date Deposited: | 25 Feb 2015 09:22 |
| Last Modified: | 25 Feb 2015 09:26 |
| URI: | http://srodev.sussex.ac.uk/id/eprint/44370 |