Expression, Purification, and Characterization of an Enzymatically Active Truncated Human Rho-Kinase I (ROCK I) Domain Expressed in Sf-9 Insect Cells

Title: Expression, Purification, and Characterization of an Enzymatically Active Truncated Human Rho-Kinase I (ROCK I) Domain Expressed in Sf-9 Insect Cells

Volume: 13 Issue: 4

Author(s): Sanjay S. Khandekar, Tracey Yi, Ed Dul, Lois L. Wrigh, Susan Chen, Gilbert F. Scott, Gary K. Smith, Dennis Lee, Erding Hu and Robert B. Kirkpatrick

Affiliation:

Keywords: ROCK I, expression, kinetics, inhibition, kinase domain

Abstract: 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.

Cite this article as:

Khandekar S. Sanjay, Yi Tracey, Dul Ed, Wrigh L. Lois, Chen Susan, Scott F. Gilbert, Smith K. Gary, Lee Dennis, Hu Erding and Kirkpatrick B. Robert, Expression, Purification, and Characterization of an Enzymatically Active Truncated Human Rho-Kinase I (ROCK I) Domain Expressed in Sf-9 Insect Cells, Protein & Peptide Letters 2006; 13(4) . https://dx.doi.org/10.2174/092986606775974357