Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting

Meier, Christoph, Brookings, Daniel C, Ceska, Thomas A, Doyle, Carl, Gong, Haiping, McMillan, David, Saville, Giles P, Mushtaq, Adeel, Knight, David, Reich, Stefanie, Pearl, Laurence H, Powell, Keith A, Savva, Renos and Allen, Rodger A (2012) Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting. Journal of Structural Biology, 177 (2). pp. 329-334. ISSN 1095-8657

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Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.

Item Type: Article
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Q Science > QD Chemistry > QD0901 Crystallography
R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology Including cancer and carcinogens
Depositing User: Laurence Pearl
Date Deposited: 30 Sep 2014 14:41
Last Modified: 02 Jul 2019 22:15

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