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Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting
journal contribution
posted on 2023-06-08, 18:34 authored by Christoph Meier, Daniel C Brookings, Thomas A Ceska, Carl Doyle, Haiping Gong, David McMillan, Giles P Saville, Adeel Mushtaq, David Knight, Stefanie Reich, Laurence PearlLaurence Pearl, Keith A Powell, Renos Savva, Rodger A AllenStructural 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.
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Publication status
- Published
File Version
- Published version
Journal
Journal of Structural BiologyISSN
1095-8657Publisher
ElsevierExternal DOI
Issue
2Volume
177Page range
329-334Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2014-09-30First Compliant Deposit (FCD) Date
2014-09-30Usage metrics
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