title: Rapid, robotic, small-scale protein production for NMR screening and structure determination.
abstract: Three-dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time-consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate. We developed a semi-automated protocol for isotopically-labeled protein production using the Maxwell-16, a commercially available bench top robot, that allows for single-step target screening by 2D NMR. In the span of a week, one person can express, purify, and screen 48 different (15)N-labeled proteins, accelerating the validation process by more than 10-fold. The yield from a single channel of the Maxwell-16 is sufficient for acquisition of a high-quality 2D (1)H-(15)N-HSQC spectrum using a 3-mm sample cell and 5-mm cryogenic NMR probe. Maxwell-16 screening of a control group of proteins reproduced previous validation results from conventional small-scale expression screening and large-scale production approaches currently employed by our structural genomics pipeline. Analysis of 18 new protein constructs identified two potential structure targets that included the second PDZ domain of human Par-3. To further demonstrate the broad utility of this production strategy, we solved the PDZ2 NMR structure using [U-(15)N,(13)C] protein prepared using the Maxwell-16. This novel semi-automated protein production protocol reduces the time and cost associated with NMR structure determination by eliminating unnecessary screening and scale-up steps.
authors.affiliation: Department of Biochemistry and Center for Eukaryotic Structural Genomics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Davin R
authors.initials: DR
authors.lastname: Jensen
authors.forename: Christopher
authors.initials: C
authors.lastname: Woytovich
authors.forename: Margie
authors.initials: M
authors.lastname: Li
authors.forename: Petar
authors.initials: P
authors.lastname: Duvnjak
authors.forename: Michael S
authors.initials: MS
authors.lastname: Cassidy
authors.forename: Ronnie O
authors.initials: RO
authors.lastname: Frederick
authors.forename: Lai F
authors.initials: LF
authors.lastname: Bergeman
authors.forename: Francis C
authors.initials: FC
authors.lastname: Peterson
authors.forename: Brian F
authors.initials: BF
authors.lastname: Volkman
mesh_terms: D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D012371:Robotics / Q000295:instrumentation* / Q000379:methods*
chemical_list: D011994:Recombinant Proteins