Day 2 | PDF Brochure

Monday, May 14

7:30 am Conference Registration and Morning Coffee

8:40 A Streamlined Optimization Process to Effectively Support Expression and Purification of Drug Target Enzymes for High-Throughput Screening
Daniele Carettoni, Head, Biochemistry, Axxam
A limiting factor in high-throughput screening (HTS) of enzymatic targets is the protein supply, which generally amounts to several milligrams of pure and functional recombinant enzymes. Optimization of the expression and purification procedures to increase protein yield is a labor-intensive and time-consuming process, which is often incompatible with the needs of drug discovery research. To overcome this bottleneck, we have developed a streamlined process for the sequential optimization of recombinant expression and purification of drug target enzymes, by applying high-throughput technologies. Each chimeric version of the target is expressed in insect cells in miniaturized 24 deep-well format. The culture conditions, cell lines, time of expression, and media additives are each varied to create a matrix of 128 alternative conditions. In a similar manner, each target protein is purified by robot-assisted affinity chromatography in 96-well format using a matrix of 24 different conditions. The detection of the relative expression level and purification yield is accomplished by a high-throughput sensitive luminescence-based readout in 96-well format. The subsequent automated optimization of the enzymatic reaction in 384-well format on a matrix of 250 different conditions further decreases the protein amount required for HTS. Data derived from HTS assays for 15 human enzymes comprising proteases, kinases, lipases and oxidoreductases will be discussed, presenting evidence that the streamlined optimization of the expression and purification conditions is often a prerequisite to support the high-throughput screening, by increasing the efficiency and quality of the drug discovery process.

9:10 Automation for Higher Throughput in Protein Expression: Visions, Facts, and Fictions
Marion Mahnke, Ph.D., NIBR Biologics Center, Novartis Pharma AG
Down-scaling, parallelization and automation are the trends in the field of recombinant protein expression in the post genomic era. During the past years many companies and academic institutions have heavily invested in process and automation technologies. Does the trend keep its promise? Can post genomic protein production issues be overcome with few automated processes? We highlight two years of experience in running a Protein Production Center in an industrial environment applying the expression systems BEVS, E. coli (and transient HEK.EBNA). We describe our streamlined and automated processes, discuss results and propose strategies to eliminate remaining bottlenecks, such as weaknesses of current expression systems and the inherent and unpredictable instability of recombinant proteins.

9:40 High-Cell Density Fermentation to Yield the High Hanging Fruits
Anne Diehl, Ph.D., NMR Supported Structural Biology, Leibnitz Institut for Molecular Pharmacology
A new fermentation system developed for high-cell mass production was adapted for the needs of recombinant protein production in E. coli. A ten-fold yield of protein/volume compared to the conventional shaking culture was achieved. The requirements of NMR-supported structural biology were fulfilled by a decrease of costs up to 1/6.

10:10 Grand Opening Refreshment Break,Poster and Exhibit Viewing 

11:10 An Overview of Parallel Protein Production at Pfizer La Jolla
Ciaran N. Cronin, Ph.D., Head, Parallel Protein Production Group, Pfizer Global R&D La Jolla
A parallel protein production platform has been established at Pizer's La Jolla, California site to support its structure-based drug design programs, and to furnish reagents for high-throughput screening, biophysical analysis and assay development. The process utilizes customized TOPO cloning vectors, plate-based microexpression screening strategies, parallel expression scale-up functions using Ultra Yield flasks for E. coli protein production and Wave bioreactors for insect cell protein production, and parallel protein purification strategies. A custom designed SQL database tracks all steps from construct design through to protein deliverables, allowing global access to experimental findings. The process has been applied successfully to provide purified protein from multiple protein families.

12:10 pm Problem Solving Roundtables

Rapid Identification of Effective Host Strains for Biopharmaceutical Production Using the Pseudo-monas-Based Pfenex Expression TechnologyTM 

Sponsored by

Dowpharma

2:30 Chairperson's Remarks

2:35 Cell-Free Overproduction of Membrane Proteins in the Presence of Detergents
Jean-Michel Betton, Ph.D., Unité Biochimie Structurale, Institut Pasteur
Analysis of genomes indicates that about one third of genes encode integral membrane proteins, but these important proteins comprise a small fraction of the entries in structural databases. A number of features of membrane proteins render them challenging targets for the structural biologist, among which the most important is the difficulty in obtaining sufficient quantities of purified protein. Recent developments in cell-free expression systems have been made to improve protein yields up to several milligrams. The open and flexible design of these in vitro systems permits a direct manipulation of the biosynthesis reactions, notably by the addition of detergent micelles. Strategies for optimizing gene expression to overproduce membrane proteins in the presence of nonionic detergents will be presented.

3:05 Protein Microarrays On-Demand
Deb Chatterjee, Ph.D., Associate Director and Senior Principal Scientist, SAIC-National Cancer Institute Frederick
Microarray technologies offer an undisputed and powerful tool to large scale analysis gene expression and polymorphisms. However, protein array technology has lagged behind DNA arrays because of various technical difficulties. Here we describe a simple protein microarray strategy wherein protein microarrays can be generated by printing expression plasmid DNAs onto slides, synthesizing target proteins in situ by cell-free protein synthesis followed by immobilization of the newly synthesized proteins through high-affinity interactions with a short nucleotide sequence embedded in the plasmid. Proof-of-concept of this technology has been demonstrated by synthesizing target proteins fused with E. coli Tus protein and immobilizing the fusion proteins onto the arrayed plasmids containing “Ter” site(s) through Tus-Ter interaction. 

3:35 Refreshment Break, Poster and Exhibit Viewing

4:15 X-Ray Crystallography of Cell-Free Synthesized Proteins
Geoffrey Chang, Ph.D., Assistant Professor, Molecular Biology, Scripps Research Institute (invited)

5:15-6:30 Networking Reception in the Exhibit Hall