Expression, purification, and characterization of an enzymatically active truncated human rho-kinase I (ROCK I) domain expressed in Sf-9 insect cells.
Rho Kinase I (ROCK I) is a serine/threonine kinase that is involved in diverse cellular signaling. To further understand the physiological role of ROCK I and to identify and develop potent and selective inhibitors of ROCK I, we have overexpressed and purified a constitutively active dimeric human ROCK I (3-543) kinase domain using the Sf9-baculovirus expression system. In addition, using a limited proteolysis technique, we have identified a minimal functional subdomain of ROCK I that can be used in crystallization studies. The availability of multimilligram amounts of purified and well characterized functional human ROCK I kinase domains will be useful in screening and structural studies.
Khandekar
Sanjay S
SS
Gene Expression and Protein Biochemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA. sanjay.khandekar@gsk.com
Yi
Tracey
T
Dul
Ed
E
Wright
Lois L
LL
Chen
Susan
S
Scott
Gilbert F
GF
Smith
Gary K
GK
Lee
Dennis
D
Hu
Erding
E
Kirkpatrick
Robert B
RB
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000494:pharmacology; D018411:Spodoptera; D019311:Staurosporine / Q000494:pharmacology; D054460:rho-Associated Kinases
D000577:Amides; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D019311:Staurosporine; C049347:fasudil