Paul G. Blommel

Enhanced Bacterial Protein Expression During Auto-Induction Obtained by Alteration of Lac Repressor Dosage and Medium Composition

The auto‐induction method of protein expression in E. coli is based on diauxic growth resulting from dynamic function of lac operon regulatory elements (lacO and LacI) in mixtures of glucose, glycerol, and lactose. The results show that successful execution of auto‐induction is strongly dependent on the plasmid promoter and repressor construction, on the oxygenation state of the culture, and on the composition of the auto‐induction medium. Thus expression hosts expressing high levels of LacI during aerobic growth exhibit reduced ability to effectively complete the auto‐induction process. Manipulation of the promoter to decrease the expression of LacI altered the preference for lactose consumption in a manner that led to increased protein expression and partially relieved the sensitivity of the auto‐induction process to the oxygenation state of the culture. Factorial design methods were used to optimize the chemically defined growth medium used for expression of two model proteins, Photinus luciferase and enhanced green fluorescent protein, including variations for production of both unlabeled and selenomethionine‐labeled samples. The optimization included studies of the expression from T7 and T7‐lacI promoter plasmids and from T5 phage promoter plasmids expressing two levels of LacI. Upon the basis of the analysis of over 500 independent expression results, combinations of optimized expression media and expression plasmids that gave protein yields of greater than 1000 μg/mL of expression culture were identified.

Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination.

AbstractThe Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsisopen reading frames into Escherichia coli expression vectors are presented along with an analysis of results from ~2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold–function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel GatewayTM protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the GatewayTM system.

High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics.

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)6-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated ÄKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)6-MBP tags by TEV protease, (His)6-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and 15N and 13C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.

Autoinduction of Protein Expression

This unit contains protocols for the use of lactose‐derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X‐ray structural studies, and multi‐well plate growth for screening and optimization are also included. Curr. Protoc. Protein Sci. 56:5.23.1‐5.23.18.

Flexi Vector Cloning

A protocol for ligation-dependent cloning using the Flexi Vector method in a 96-well format is described. The complete protocol includes PCR amplification of the desired gene to append Flexi Vector cloning sequences, restriction digestion of the PCR products, ligation of the digested PCR products into a similarly digested acceptor vector, transformation and growth of host cells, analysis of the transformed clones, and storage of a sequence-verified clone. The protocol also includes transfer of the sequence-verified clones into another Flexi Vector plasmid backbone. Smaller numbers of cloning reactions can be undertaken by appropriate scaling of the indicated reaction volumes.

Crystal structure of At2g03760, a putative steroid sulfotransferase from Arabidopsis thaliana

David W. Smith, Kenneth A. Johnson, Craig A. Bingman, David J. Aceti, Paul G. Blommel, Russell L. Wrobel, Ronnie O. Frederick, Qin Zhao, Hassan Sreenath, Brian G. Fox, Brian F. Volkman, Won Bae Jeon, Craig S. Newman, Eldon L. Ulrich, Adrian D. Hegeman, Todd Kimball, Sandy Thao, Michael R. Sussman, John L. Markley, and George N. Phillips, Jr.* Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin Center for Eukaryotic Structural Genomics, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin

Multiplexing Fluorescence Polarization Assays to Increase Information Content Per Screen: Applications for Screening Steroid Hormone Receptors

As the push to reduce cost per well in high-throughput screening reaches the practical limitations of liquid handling, future cost savings will likely arise from an increase in information content per well. One strategy to increase information content is to perform discreet assays against multiple targets in a single well. In such assays, reagent usage and liquid handling steps do not scale-up in direct proportion to the increase in information content, providing for a simple method to increase data points per screen without further reductions in assay volume. The authors have used tracers incorporating the spectrally distinct fluorophores fluorescein and TAMRA to develop a high-throughput assay to identify selective estrogen receptor α or proges-terone receptor ligands. Selectivity is assessed immediately in this assay, with no requirement for separate follow-up screening to determine selectivity. This methodology is easily adaptable to other target classes.

Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies

Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or 1H–15N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.

X‐ray structure of Arabidopsis At1g77680, 12‐oxophytodienoate reductase isoform 1

Brian G. Fox,* Thomas E. Malone, Kenneth A. Johnson, Stacey E. Madson, David Aceti, Craig A. Bingman, Paul G. Blommel, Blake Buchan, Brendan Burns, John Cao, Claudia Cornilescu, Jurgen Doreleijers, Jason Ellefson, Ronnie Frederick, Holokere Geetha, David Hruby, Won Bae Jeon, Todd Kimball, John Kunert, John L. Markley, Craig Newman, Andrew Olson, Francis C. Peterson, George N. Phillips Jr., John Primm, Bryan Ramirez, Nathan S. Rosenberg, Mike Runnels, Kory Seder, Jeff Shaw, David W. Smith, Hassan Sreenath, Jikui Song, Michael R. Sussman, Sandy Thao, Donna Troestler, Ejan Tyler, Robert Tyler, Eldon Ulrich, Dimitriy Vinarov, Frank Vojtik, Brian F. Volkman, Gary Wesenberg, Russell L. Wrobel, Jie Zhang, Qin Zhao, and Zolt Zolnai University of Wisconsin Center for Eukaryotic Structural Genomics, University of Wisconsin–Madison, Madison, Wisconsin Molecular and Environmental Toxicology Program, University of Wisconsin–Madison, Madison, Wisconsin Biophysics Doctoral Program, University of Wisconsin–Madison, Madison, Wisconsin

Crystal structure of gene locus At3g16990 from Arabidopsis thaliana

The Center for Eukaryotic StructuralGenomicsisdedicatedtodeterminingthestructuresofnovelproteins from eukaryotic organisms. Open reading framesare scored using thirteen different categories (i.e. new foldprediction, solubility prediction, small percentage of lowcomplexity sequence, etc.) and then ranked to indicate theirsuitabilityforstudybynuclearmagneticresonance(NMR)orX-raycrystallography.GenelocusAt3g16990from