Engineering human MEK-1 for structural studies: a case study of combinatorial domain hunting
Meier, C. and Brookings, D.C. and Ceska, T.A. and Doyle, C. and Gong, H. and McMillan, D. and Saville, G.P. and Mushtaq, A. and Knight, D. and Reich, S. and Pearl, L.H. and Powell, K.A. and Savva, Renos and Allen, R.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 1047-8477.
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.
|Keyword(s) / Subject(s):||Gene fragmentation, Protein expression, Structural biology, MEK-1, Domain hunting|
|School:||Birkbeck Schools and Departments > School of Science > Biological Sciences|
|Research Centre:||Structural Molecular Biology, Institute of (ISMB), Innovation Management Research, Birkbeck Centre for|
|Date Deposited:||09 Feb 2012 13:56|
|Last Modified:||06 Dec 2016 10:44|
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