High yield expression of non-phosphorylated protein tyrosine kinases in insect cells.
The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.
Wang
Leyu
L
Research Technology Center, Pfizer Cambridge Laboratories, 620 Memorial Drive, Cambridge, MA 02139, USA. Leyu.Wang@pfizer.com
Foster
Meta
M
Zhang
Yan
Y
Tschantz
William R
WR
Yang
Lily
L
Worrall
Joe
J
Loh
Christine
C
Xu
Xu
X
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D006801:Humans; D053616:Janus Kinase 3 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D036104:Receptor, EphA2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism* / Q000821:virology
D001425:Bacterial Outer Membrane Proteins; D011505:Protein-Tyrosine Kinases; D036104:Receptor, EphA2; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D053616:Janus Kinase 3; D017027:Protein Tyrosine Phosphatases; C067571:yopH protein, Yersinia