Protein Expression Plasmids Produced Rapidly: Streamlining Cloning Protocols and Robotic Handling

• DOI: 10.1089/adt.2005.3.661
• Publication Date: 2006-01-26T12:07:31Z
• Authors: Maria Kornienko; Allison Montalvo; Brian E. Carpenter; Michael Lenard; Pravien Abeywickrema; Dawn L. Hall; Paul L. Darke; Lawrence C. Kuo
• Keywords: 0301 basic medicine; rho-Associated Kinases; 0303 health sciences; Time Factors; Protein Conformation; Drug Evaluation, Preclinical; Intracellular Signaling Peptides and Proteins; Robotics; Protein Serine-Threonine Kinases; Polymerase Chain Reaction; Recombinant Proteins; 3. Good health; Automation; 03 medical and health sciences; Mutagenesis; Cloning, Molecular; Crystallization; Software; Plasmids

As many processes in the preclinical drug discovery process become highly parallel, the need to also produce a large number of different proteins in parallel has become acute, such as for protein crystallization and activity screening. In turn, the requisite DNA constructions to produce these proteins must now be done at a rate that requires automated cloning procedures, each with an intrinsic low failure probability per sample. The high-throughput cloning solutions presented here achieve production of 192 different expression plasmids at a success rate of greater than 95% of the targeted open reading frames. Time for completion of the set by one person is reduced to approximately 11 working days, starting with polymerase chain reactions for a number of source clones and ending with purified expression plasmids. Achievement of this throughput utilizes the following: (1) the Beckman Coulter (Fullerton, CA) Biomek FX liquid handler for most manipulations, (2) Gateway cloning technology (Invitrogen Corp., Carlsbad, CA), and (3) computer programs designed for parallel processing of all sample information, including primer design and the resulting DNA and protein sequence assembly. Exemplary data are presented for discovery of a form of the Rho-kinase that crystallizes (ROCK2).