Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting
| Autor: | David McMillan, Laurence H. Pearl, Keith A Powell, Carl Brendan Doyle, Thomas A Ceska, Giles P Saville, Adeel Mushtaq, Stefanie Reich, Renos Savva, Rodger A. Allen, Daniel Christopher Brookings, David Knight, Haiping Gong, Christoph Meier |
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| Rok vydání: | 2012 |
| Předmět: |
Genetics
Crystallography MAP Kinase Kinase 1 Computational biology Biology Protein Engineering Peptide Fragments Recombinant Proteins Protein Structure Tertiary Market fragmentation Domain (software engineering) Structural biology Fragment (logic) Protein kinase domain Structural Biology Escherichia coli Humans Cloning Molecular Experimental methods Crystallization Gene Biological sciences |
| Zdroj: | Journal of Structural Biology. 177:329-334 |
| ISSN: | 1047-8477 |
| DOI: | 10.1016/j.jsb.2012.01.002 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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