title: Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting.
abstract: 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.
authors.affiliation: UCB Pharma, 216 Bath Road, Slough SL1 4EN, United Kingdom. chris.meier@ucb.com
authors.forename: Christoph
authors.initials: C
authors.lastname: Meier
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Brookings
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Ceska
authors.forename: Carl
authors.initials: C
authors.lastname: Doyle
authors.forename: Haiping
authors.initials: H
authors.lastname: Gong
authors.forename: David
authors.initials: D
authors.lastname: McMillan
authors.forename: Giles P
authors.initials: GP
authors.lastname: Saville
authors.forename: Adeel
authors.initials: A
authors.lastname: Mushtaq
authors.forename: David
authors.initials: D
authors.lastname: Knight
authors.forename: Stefanie
authors.initials: S
authors.lastname: Reich
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Pearl
authors.forename: Keith A
authors.initials: KA
authors.lastname: Powell
authors.forename: Renos
authors.initials: R
authors.lastname: Savva
authors.forename: Rodger A
authors.initials: RA
authors.lastname: Allen
mesh_terms: D003001:Cloning, Molecular; D003460:Crystallization; D003461:Crystallography; D004926:Escherichia coli; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000737:chemistry* / Q000235:genetics*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human