Faculty Collaboration Database

Rapid, robotic, small-scale protein production for NMR screening and structure determination. Protein Sci 2010 Mar;19(3):570-8

Date

01/15/2010

Pubmed ID

20073081

Pubmed Central ID

PMC2866281

DOI

10.1002/pro.335

Scopus ID

2-s2.0-77249156883   6 Citations

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.

Author List

Jensen DR, Woytovich C, Li M, Duvnjak P, Cassidy MS, Frederick RO, Bergeman LF, Peterson FC, Volkman BF

Authors

Petar Duvnjak MD Assistant Professor in the Radiology department at Medical College of Wisconsin
Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of Wisconsin
Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Humans
Nuclear Magnetic Resonance, Biomolecular
Protein Biosynthesis
Protein Conformation
Recombinant Proteins
Robotics