Achieving higher throughput protein processing requires an arsenal of techniques and supporting technologies to streamline steps, ensure quality and reach desired results more quickly. This meeting will explore the strategies used to optimize purification within the context of HTP, while examining options for monitoring protein processing and analyzing results to shed light on proteins’ condition and biological activity. Continually gathering and organizing data will also be addressed, including software options and LIMS. Additional insights will be presented for clone selection, optimizing buffers, and removing tags. Automating throughput, such as liquid handling systems and microfluidics, will be discussed, along with implementing single-use and disposable technologies.
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TUESDAY, JANUARY 10
1:30 pm Conference Registration
2:00 BuzZ Session A
2:45 Networking Refreshment Break in the Exhibit Hal, Poster Awards
3:30 BuzZ Session B
4:15 End of Day
4:30 – 7:30 Concurrent Dinner Short Courses (SC5-SC9)
*Separate Registration Required.
WEDNESDAY, JANUARY 11
7:00 am Conference Registration
7:30 Breakfast Presentation (Sponsorship Opportunity Available) or Morning Coffee
8:15 Chairperson’s Opening Remarks
Opher Gileadi, Ph.D., Principal Investigator, Biotechnology and Genomic Integrity, The Structural Genomics Consortium (SGC), University of Oxford
» KEYNOTE PRESENTATION
8:20 High-Throughput Screening for Chromatographic and Non-Chromatographic Protein Purification Methods
Steven Cramer, Ph.D., William Weightman Walker Professor, Chemical and Biological Engineering, Rensselaer Polytechnic Institute - Biography
This talk will present examples of high-throughput screening for the development of novel chromatographic and non-chromatographic methods. Parallel batch robotic adsorption experiments are employed to identify high affinity and selective displacers for ion exchange systems, and unique selectivity windows in hydroxyapatite and multimodal chromatographic systems. In addition, high-throughput screening is used to evaluate a range of operating conditions for the optimization and development of robust smart biopolymer affinity precipitation systems.
9:00 High-Throughput Cloning, Expression and Purification: An Industry Perspective
Andrew Fosberry, Ph.D., Manager, Expression and Fermentation Sciences, GlaxoSmithKline
The expression and purification of proteins for high throughput screening and crystallography was historically performed in a low throughput mode on a protein-by-protein basis. Production of active, high purity material on a small number of selected proteins involved time-consuming, reiterative approaches to expression: re-design of constructs, moving to more complex expression systems and using novel purification strategies until a successful outcome was achieved. To transform this process into a truly high throughput, and ultimately automated procedure required a complete rethinking of the philosophy and methodology of expression and purification. This presentation will attempt to address the issues and feasibility of a truly high throughput expression and purification process, highlighting the bottlenecks that arose using these technologies, key learning’s and where we are now.
9:30 Beyond the Plan, What Really Happens in an HTP Project: Case Studies from a Core Service Lab
William Gillette, Ph.D., Senior Scientist, Protein Expression Lab, SAIC-Frederick, Inc. - Biography
Using several case studies, I will discuss the pitfalls and practical issues surrounding high-throughput protein purification in the context of a core service lab. Emphasis will be on the purification process, micro-scale screening, scale-up from micro-scale (micro-liters) to midi-scale (liters), data acquisition and managing the entire process.
10:00 Networking Coffee Break in the Exhibit Hall, Poster Viewing
10:45 Test Method Development for High-Throughput Processes
Steven S. Kuwahara, Ph.D., Principal, QC, GXP Biotechnology, LLC - Biography
Planning ahead is critical for developing test methods for use with a high-throughput process. Test methods that are satisfactory for testing raw material and final products are often unsuitable when employed in a high-throughput setting. When properly planned, it is possible to take advantage of the principles of process analytical technology (PAT) to compensate for the problems caused by the need for quick test methods. We will discuss the factors that must be considered when developing test methods for working in this environment.
11:15 Human Topoisomerase II α: High-Yield Production of Biologically Active Protein and Development of a High-Throughput Assay
Andrew J. Theobald, Investigator, Biological Reagents & Assay Development, GlaxoSmithKline - Biography
DNA topoisomerase type II enzymes are well-validated targets for the treatment of cancer and bacterial infection, and human topoisomerase IIα in vitro assays play an important role in supporting drug discovery efforts. We have developed a high-yield method for the rapid over-expression and purification of GST-tagged human topoisomerase IIα from baculovirus infected insect cells. The enzyme has been shown to be functionally active, demonstrating ATP-dependent relaxation of supercoiled DNA in an agarose gel-based assay. In addition we have demonstrated a DNA-dependent increase in ATPase activity in a high-throughput fluorescence assay, and obtained IC50 values for known inhibitors in good agreement with published data. This validated source of enzyme and high-throughput assay provides valuable tools for the identification of new inhibitors and essential selectivity studies.
11:45 Protein Arrays for Assessment of Human Anti-Viral Antibody Responses
Jonathan S. Duke-Cohan, Ph.D., Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Institutes of Medicine - Biography
Protein arrays offer a rapid means for testing the human antibody response targeted against viral antigens, correlating the identified specificities with protection, and using this information to design recombinant vaccine protocols. High-throughput protein arrays, however, are only an initial step as folding, post-translational modifications, and even protein packing on the viral membrane exert a powerful influence over immunogenicity. Medium throughput arrays utilizing a focused subset of high quality targets offer a means of bridging the potential gap between high-throughput arrays expressing reduced quality proteins and real-world significance.
12:15 pm Close of Morning Session
12:30 Luncheon Presentation I
The Gel-Free, Blot-Free, Hands-Free Simple Western is Here!
Peter Fung, Ph.D., Product Manager, ProteinSimple
Simple Western assays run on Simon reinvent the entire Western blotting process, finally giving researchers a completely automated, walk-away solution. The reproducibility and quantitation challenges as well as time to result bottlenecks commonly experienced with traditional Westerns are easily addressed. Throughput benefits and applications of the Simple Western will be discussed.
1:00 Luncheon Presentation II (Sponsorship Opportunity Available)
2:00 Chairperson’s Remarks
William Gillette, Ph.D., Senior Scientist, Protein Expression Lab, SAIC-Frederick, Inc.
2:05 Development of a Structural Proteomics Pipeline - Automation, Integration and Data Management
Ray Owens, Ph.D., NDM Senior Research Fellow, Division of Structural Biology, Oxford Protein Production Facility, University of Oxford - Biography
The Oxford Protein Production Facility (OPPF) was among the first structural proteomics projects in Europe and has now been running for nearly ten years. We have focused on a combination of E. coli and mammalian-based expression systems for protein production and have developed common platforms for protein purification and quality assessment. Running a structural proteomics pipeline presents a number of challenges including how best to integrate the different processes involved and how to keep track of the relatively large amounts of data that are generated. Experience gained, lessons learnt and current developments in the OPPF will be discussed.
2:35 Automating Protein Purification for Genomic Enzymology Research
Sirano Dhe-Paganon, Ph.D., Assistant Professor, Physiology, University of Toronto - Biography
A resource-intensive step in genomic enzymology research is the production of many pure proteins in the milligram-scale range. Intracellular protein purification from recombinant bacteria has required as a first step the separation of culture medium from cell mass and the separation of cell debris from lysed cells. Modern IMAC beads allowed the development of a column and nozzle assembly that can be used in-line with high-pressure and high-flow microfluidization for the purification of bacterially-overexpressed, His-tagged proteins directly from bacterial cultures. Yields and purity are comparable with standard protocols. This large-scale protein purification tool is easy to use and widely applicable.
3:05 Rapid Optimization of Purification Conditions Using an Automated Small-Scale Purification Platform
Ruth Steele, Ph.D., Research Scientist, Structural Biology, US C.R.E.A.Te, Johnson & Johnson Pharmaceutical R&D
The optimization of purification conditions for difficult to purify targets can be resource intensive. We have a developed an automated protein purification platform to rapidly and screen column media and buffers to optimize purification conditions. The ASAP (Atoll Small-scale Automated Purification) platform integrates four essential element; complex buffer array preparation, small-scale parallelized purification, automated liquid handling and data analysis. The integration of this robotic purification platform will be discussed along with some example data.
3:35 POSTER HIGHLIGHT
High Throughput Protein Purification Setup at the Novo Nordisk Foundation Center for Protein Research, Poster B212
Tine Kragh Nielsen, Team Leader, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen
3:50 Networking Refreshment Break
4:30 High-Throughput Expression and Purification of Membrane Proteins
Filippo Mancia, Ph.D., Assistant Professor, Physiology & Cellular Biophysics, Columbia University
High-resolution structure determination relies on the ability of the macromolecule to form ordered crystals that diffract X-rays. While crystallization remains somewhat empirical, for a given protein, success is proportional to the number of conditions screened and to the number of variants tried. Success rate for membrane proteins is substantially lower than their soluble counterparts, emphasizing the importance of a robust screening platform to identify those well-expressing targets with increased probability of crystallization. Presented here is one such platform for expression and detergent stability screening of recombinant prokaryotic membrane proteins over-produced in Escherichia coli.
5:00 Functional Analysis of Proteases from S. pneumoniae, B. anthracis, and Y. pestis
Scott Peterson, Ph.D., Professor , Director, Functional Genomics Research Technology, J. Craig Venter Institute - Biography
A set of 187 protease candidates were mined from three pathogenic bacteria, Streptococcus pneumoniae TIGR4, Bacillus anthracis Ames and Yersinia pestis KIM. We assessed the utility of various fusion tags including: hexa-histidine (His-tag), maltose binding protein (MBP), MBP with signal peptide (SP-MBP), disulfide oxydoreductase A (DsbA) and glutathione S-transferase (GST). A large fracton of these candidates were recovered (86.1%). Protease activities of the purified proteins were examined using a high throughput fluorescence-based assay and zymography, that not only support the annotation as a protease or putative protease but also identified the protease activity of hypothetical proteins with no previous association with protease activity.
5:30 - 6:30 Reception in the Exhibit Hall with Poster Viewing
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Links to Companion Meetings
Protein Aggregation and Emerging Analytical Tools
Protein Purification & Recovery