Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase.
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.
Qian
Kevin C
KC
Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals Inc., Research and Development, Ridgefield, CT 06877, USA. kqian@rdg.boehringer-ingelheim.com
Studts
Joey
J
Wang
Lian
L
Barringer
Kevin
K
Kronkaitis
Anthony
A
Peng
Charline
C
Baptiste
Alistair
A
LaFrance
Roger
R
Mische
Sheenah
S
Farmer
Bennett
B
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
D001051:Apoenzymes; D017931:DNA Primers; D011994:Recombinant Proteins; D051573:Proto-Oncogene Proteins c-pim-1