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Combinatorial domain hunting: an effective approach for the identification of soluble protein domains adaptable to high-throughput applications

journal contribution
posted on 2023-06-08, 14:43 authored by Stephanie Reich, Loretto H Puckey, Caroline L Cheetham, Richard Harris, Ammar A E Ali, Uma Bhattacharyya, Kate Maclagan, Keith A Powell, Chrisostomos ProdromouChrisostomos Prodromou, Laurence PearlLaurence Pearl, Paul C Driscoll, Renos Savva
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.

History

Publication status

  • Published

Journal

Protein Science

ISSN

0961-8368

Publisher

Wiley-Blackwell

Issue

10

Volume

15

Page range

2356-2365

Department affiliated with

  • Biochemistry Publications

Full text available

  • No

Peer reviewed?

  • Yes

Legacy Posted Date

2015-02-25

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