Archived Content
PXEB 09 Header
Co-Located with BIOTECHNICA 2009, Europe's No.1 Event in Biotechnology and Life Sciences 

Day 1 |  Day 2


THURSDAY, 8 OCTOBER

 

Utilizing Next Generation Methods
and Technologies

09:00-09:05 Chairperson’s Remarks

Julio Camarero, Ph.D., Associate Professor, Pharmaceutical Sciences and Toxicology, School of Pharmacy, University of Southern California

09:05-09:35 Transgene Expression via Modifications of the Coding Sequence

Asli Bauer, Ph.D., Senior Scientist, Molecular Microbiology and Gene Therapy, University of Regensburg

The efficacy of transgene expression is significantly enhanced by RNA and codon optimization. A decrease of CpG content in the ORF, applied to circumvent epigenetic mechanisms of gene silencing, results in extremely low expression yields. Multi parameter RNA- and codon optimization results in enhanced de novo-synthesis of gfp and epo RNA in the nucleus and in the cytoplasm. Furthermore, RNA- and codon optimization lead to a dramatically prolonged gene expression in vivo.

09:35-10:05 Interfering Peptides Targeting Extended Protein Interaction Surfaces

Katja Arndt, Ph.D., Principle Investigator, Freiburg Institute for Advanced Studies, School of Life Sciences, University of Freiburg

Protein-protein interactions play important roles in numerous diseases, yet, targeting extended protein interaction surfaces still remains a huge challenge for today’s antibody and protein engineering. We use semi-rational design in combination with selection systems to generate peptides interfering with transcription factor-mediated gene expression. Phage display selection was compared to in vivo selection based on protein-fragment complementation assay, and both methods succeeded in selecting specific peptide inhibitors with high stability. Implementing competitive and negative design aspects resulted in major energetic differences between desired and non-desired states, thereby allowing simultaneous selection for affinity and specificity. Different strategies will be discussed for targeting the oncoproteins Myc, Jun and Fos using tailored D- and L-peptides.

10:05-10:35 Sponsored Presentation (Opportunity Available)

10:35-11:00 Coffee Break

11:00-11:30 Creating New Enzymes: High-Throughput Protein Expression Optimization of Monomeric TIM Libraries using EnBase™ Technology

Mari Ylianttila, Ph.D., Post Doctoral Fellow, Department of Process and Environmental Engineering, University of Oulu

Our aim is to build a platform of TIM variants (Kealases) with a widened substrate range based on monomeric Triosephosphate Isomerase. In order to screen for active enzymes with novel reactions and to optimize the process for recombinant protein production we utilize the EnBaseTM technology to set up a high-throughput for process optimization and for high-throughput crystallization of the mutants. EnBaseTM enables a fed-batch like cultivation in small scale, from 120 µl up to 1 L. The method is based on the enzymatic release of substrate from a gel at the bottom of the growth vessel (e.g. microtiter plate). Due to the controllable growth rate in the EnBaseTM system the cultivation mimics a large fed-batch process. High cell densities ensure high protein yields; slow growth rates ensure high amounts of soluble protein per cell.

11:30-12:00 Biosynthesis of Folded Cyclotides inside Living Bacterial Cells: A Convenient Route for Generation of Genetically-Encoded Cyclotide-Based Libraries

Julio Camarero, Ph.D., Associate Professor, Pharmaceutical Sciences and Toxicology, School of Pharmacy, University of Southern California

The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cylotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

12:00-12:30 Protein Folding on an Industrial Scale

Andreas Anton, Ph.D., Director of Process Development, Scil Proteins GmbH

The production of recombinant proteins (e.g. therapeutic proteins) in bacteria (Escherichia coli) is one of the challenging fields of biotechnology. The formation of inclusion bodies is a frequent result of high-level protein production in the cytoplasm. As a consequence the development of in vitro folding procedures is one of the mayor issues in “E. coli” production processes.  Topic of two case studies is the optimization of volume, folding time and yield of an in vitro folding step during process development. Thereby lab scale processes can be applied to industrial scale also if they use in vitro folding as one purification step. Finally competent folding development results in significantly reduction of costs for industrial processes.

12:30-13:45 Lunch for Purchase in the Exhibit Hall and Exhibit Viewing

 

Applications

14:00-14:05 Chairperson’s Remarks

Stefan Dübel, Ph.D., Director, Biotechnology, Technische Universität Braunschweig

14:05-14:35 Engineering Enzymes for Prodrug Activation Therapy

Kristian Müller, Junior Professor, Department of Biology, University of Freiburg

Tailored enzymes for tumor targeting facilitating prodrug activation therapies will enable the combination of the best properties of biologics and small molecules in highly effective approaches. We optimized key properties of the enzyme TEM beta-lactamase. Using a perturbation-compensation approach in combination with directed evolution based on NExT DNA shuffling, we raised the half-life time in a protease assay 13-fold, the kcat in a heat stress test at 60°C from zero to over 1000/s and the melting temperature over 28°C while maintaining the catalytic substrate spectrum and activity at low temperatures. We generated fusions with scFv antibody fragments and receptor ligands, addressed the problem of immunogenicity, and also optimized the split enzyme.

14:35-15:05 cGMP Production of Therapeutic Antibody-Cytokine Fusion Proteins

Leonardo Giovannini, Ph.D., Head, Protein Production, PHILOGEN S.p.A.

The complete cGMP production flow of an antibody-cytokine fusion protein for clinical use will be discussed with specific focus on clone selection, master and working cell bank preparation, fermentation, protein purification and final filling.

15:05-15:35 Sponsored Presentation (Opportunity Available)

15:35-16:00 Refreshment Break

16:00-16:30 Going Double-Digit with Pichia: High-Level Expression of Human Serum Albumin and Transferrin as Well as Fusion Proteins Driven by AOX1 Promoter Library

Roland Weis, Head, Research & Development, Biotechnology,VTU Technology

Although Pichia pastoris nowadays is a state-of-the-art host for recombinant expression with extraordinary capabilities for the secretion of heterologous proteins, only few examples of expression titers as high as >5g/L are reported. We report recombinant protein yields exceeding 10 g/L employing our proprietary AOX1-promoter library for expression of human serum albumin, human serum transferring(s) as well as fusion proteins thereof. Different cloning and expression strategies were assessed to improve expression titers, and parameter scouting in 1L bioreactors resulted in an optimized fermentation protocol, executed in 5L fermentations. As a result of downstreaming approaches, high purity and quality of the described proteins was achieved. Relying on the AOX1-promoter library, several other (human) recombinant proteins were expressed to titers between 2 and 7 g/L.

16:30-17:00 “Proteome Scale” in vitro Antibody Selections: Lessons from Pilot Projects and Use of the Antibody Genes for Functional Genomics

Stefan Dübel, Ph.D., Director, Biotechnology, Technische Universität Braunschweig

Started by work within the NGFN SMP Antibody Factory, a highly integrated and MTP- based pipeline for the generation of antibodies to any antigen was established and valiDated by making binders to individual SH2 domains within a Pilot Project of the Structural Genomics Consortium (SGC). We have further used various scFv genes for the functional analysis of the target gene. After subcloning into a mammalian expression vector, the scFv genes induced a functional knock down of the target antigen.

17:00-17:30 Automated Parallel Protein Chromatography in a
96-Array Format

Juergen Friedle, Ph.D., Senior Vice President, Marketing, Atoll GmbH

This presentation will discuss innovative technology for parallel chromatography used in resin and method screening, process analytics and depletion of abundant components. This method utilized 8 independent columns operating simultaneously, compression packed with any available resin. It is suited to process a large number of samples and fully automated. Results are available in days instead of months.

17:30 Close of Next Generation Technologies for Protein Expression conference

 


For more information, please contact:
Elizabeth Lamb, Senior Conference Director
Cambridge Healthtech Institute
E-mail: elamb@healthtech.com 

For exhibit and sponsorship information, please contact:
Carol Dinerstein
Cambridge Healthtech Institute
Email: dinerstein@healthtech.com 
Phone: 781-972-5471