Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications

Autor: Paul C. Driscoll, Stephanie Reich, Caroline L Cheetham, Renos Savva, Uma Bhattacharyya, Ammar A E Ali, Laurence H. Pearl, Richard Harris, Loretto H Puckey, Keith A Powell, Chrisostomos Prodromou, Kate Maclagan
Rok vydání: 2006
Předmět:
Zdroj: Protein Science. 15:2356-2365
ISSN: 1469-896X
0961-8368
Popis: 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.
Databáze: OpenAIRE
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