title: Protein expression plasmids produced rapidly: streamlining cloning protocols and robotic handling.
abstract: 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).
authors.affiliation: Department of Structural Biology, Merck Research Laboratories, West Point, PA 19486, USA. maria.kornienko@merck.com
authors.forename: Maria
authors.initials: M
authors.lastname: Kornienko
authors.forename: Allison
authors.initials: A
authors.lastname: Montalvo
authors.forename: Brian E
authors.initials: BE
authors.lastname: Carpenter
authors.forename: Michael
authors.initials: M
authors.lastname: Lenard
authors.forename: Pravien
authors.initials: P
authors.lastname: Abeywickrema
authors.forename: Dawn L
authors.initials: DL
authors.lastname: Hall
authors.forename: Paul L
authors.initials: PL
authors.lastname: Darke
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Kuo
mesh_terms: D001331:Automation; D003001:Cloning, Molecular / Q000379:methods*; D003460:Crystallization; D004353:Drug Evaluation, Preclinical / Q000379:methods; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics; D016296:Mutagenesis; D010957:Plasmids / Q000096:biosynthesis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012371:Robotics / Q000295:instrumentation; D012984:Software; D013997:Time Factors; D054460:rho-Associated Kinases
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases