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- Title
Protein Expression Plasmids Produced Rapidly: StreamliningCloning Protocols and Robotic Handling.
- Authors
Maria Kornienko; Allison Montalvo; Brian E. Carpenter; Michael Lenard; Pravien Abeywickrema; Dawn L. Hall; Paul L. Darke; Lawrence C. Kuo
- Abstract
As many processes in the preclinical drug discovery process become highly parallel, theneed to also produce a large number of different proteins in parallel has become acute, such as forprotein crystallization and activity screening. In turn, the requisite DNA constructions to producethese proteins must now be done at a rate that requires automated cloning procedures, each with anintrinsic low failure probability per sample. The high-throughput cloning solutions presented hereachieve production of 192 different expression plasmids at a success rate of greater than 95% of thetargeted open reading frames. Time for completion of the set by one person is reduced toapproximately 11 working days, starting with polymerase chain reactions for a number of sourceclones and ending with purified expression plasmids. Achievement of this throughput utilizes thefollowing: (1) the Beckman Coulter (Fullerton, CA) Biomek® FX liquid handler for mostmanipulations, (2) GatewayTM cloning technology (Invitrogen Corp., Carlsbad, CA), and (3)computer programs designed for parallel processing of all sample information, including primerdesign and the resulting DNA and protein sequence assembly. Exemplary data are presented fordiscovery of a form of the Rho-kinase that crystallizes (ROCK2).
- Publication
Assay & Drug Development Technologies, 2005, Vol 3, Issue 6, p661
- ISSN
1540-658X
- Publication type
Article