title: Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase.
abstract: Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1-313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14-313) by thrombin digestion during purification. The Pim-1 (14-313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 A resolution and belong to space group P6(5), with unit-cell parameters a = b = 95.9, c = 80.0 A, beta = 120 degrees and one molecule per asymmetric unit.
authors.affiliation: Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals Inc., Research and Development, Ridgefield, CT 06877, USA. kqian@rdg.boehringer-ingelheim.com
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Qian
authors.forename: Joey
authors.initials: J
authors.lastname: Studts
authors.forename: Lian
authors.initials: L
authors.lastname: Wang
authors.forename: Kevin
authors.initials: K
authors.lastname: Barringer
authors.forename: Anthony
authors.initials: A
authors.lastname: Kronkaitis
authors.forename: Charline
authors.initials: C
authors.lastname: Peng
authors.forename: Alistair
authors.initials: A
authors.lastname: Baptiste
authors.forename: Roger
authors.initials: R
authors.lastname: LaFrance
authors.forename: Sheenah
authors.initials: S
authors.lastname: Mische
authors.forename: Bennett
authors.initials: B
authors.lastname: Farmer
mesh_terms: D001051:Apoenzymes / Q000737:chemistry; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006801:Humans; D011487:Protein Conformation; D051573:Proto-Oncogene Proteins c-pim-1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
chemical_list: D001051:Apoenzymes; D017931:DNA Primers; D011994:Recombinant Proteins; D051573:Proto-Oncogene Proteins c-pim-1