PepTalk 2017
PepTalk 2017
Archived Content

Engineering Genes Vectors Constructs and Clones

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Sponsored by
DNA 2.0
7:30 am Breakfast Presentation

Maximizing Recombinant Protein Expression Through Systematic Gene Design

Mark Welch, Director of Gene Design, DNA2.0

DNA sequences are now far more readily available in silico than as physical DNA. De novo gene synthesis is an increasingly cost-effective method for building genetic constructs, and effectively removes the constraint of basing constructs on extant sequences. Here we present how gene design variables such as codon choice and mRNA structure predictably affect the yield of recombinant protein expression, often up to orders of magnitude increase in expression levels. Host systems validated include mammalian cell lines (CHO/HEK293), yeast (S.cerevisiae/P.pastoris/K.lactis), E.coli and more.


Engineering Expression Systems

8:15 Chairperson's Opening Remarks
Charles Squires, Ph.D., VP of Discovery and Partnerships, Pfenex Inc.

8:20 Engineering and Evaluation of Mammalian Secreted Protein Expression Library for High-Throughput Target Discovery

Tadas Panavas, Ph.D., Senior Scientist, Molecular and Protein Biosciences, Centocor R&D, Inc.

Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We engineered a vector system where from the same plasmid it was possible to express either a native or tagged protein. We optimized small scale 96-well format mammalian expression in HEK293E cells, and evaluated this vector-expression system using ~1500 secreted proteins.

8:50 Engineering Protein Glycosylation Pathways in the Baculovirus-Insect Cell System

Donald Jarvis, Ph.D., Professor, Molecular Biology, University of Wyoming

The protein glycosylation pathways of the insect cell lines used as hosts for baculovirus expression vectors are simpler than those of higher eukaryotes and, as a result, the baculovirus-insect cell system is unable to produce sialylated glycoproteins. This is a significant problem because insect glycosylation patterns are not always compatible with recombinant glycoprotein applications. However, this problem can be addressed using glycoengineering approaches, which have been developed for the baculovirus-insect cell system over the past 15 years. In essence, this approach involves transforming insect cell lines with constitutively expressable genes encoding higher eukaryotic protein glycosylation functions. The addition of these transgenes can give rise to functionally stable, genetically improved insect cell lines capable of providing human-type glycosylation of baculovirus-expressed recombinant glycoproteins.

Sponsored by
VTU Technology
9:20 Driving a Variant of the Methanol-Inducible AOX1 Promoter from Pichia pastoris with Glycerol for High-Level Expression of (Therapeutic) Proteins

Roland Weis, Ph.D., Head of Operations, VTU Technology GmbH, Austria

With its high secretory capacity, Pichia pastoris is ideally suited for high-level expression of heterologous proteins for therapeutic or industrial usage. VTU Technology´s unique PAOX1 promoter library controls the expression of target gene(s) under two applicable cultivation regimes, induction by methanol feed as well as by derepression under glycerol feed. A cultivation capacity of >20.000 clones per week in 96-deep well plates enables the development of customized high performance expression strains at unrivaled speed.

Sponsored by
Delphi Genetics 
9:50 Developing Effective Technologies using Bacterial Poison-Antidote Genes for the Production of Recombinant Protein in E. coli
Philippe Gabant, Ph.D., Head of Business Development, Delpi Genetics SA
We have developed an innovative platform (strains and vectors) for the expression of recombinant protein, our innovation is focused on the use of natural bacterial poison and antidote genes for the selection of expression vectors.   Our design of the poison and antidote genes allows to work without antibiotics and antibiotic markers on the vector.  Industrial partners have shown that this StabyExpress technology is not only interesting from a regulatory standpoint (removal of antibiotics) it also greatly enhanced the yield of recombinant protein production.

10:05 Networking Coffee Break in the Exhibit Hall with Poster Viewing

10:45 Influence of Histidine Affinity Tags on Protein Structural Order

Evelina Angov, M.S., Ph.D., Microbiologist, Malaria Vaccine Development, Walter Reed Army Institute of Research

The incorporation of affinity tags into cloned sequences has greatly facilitated purification process development for protein antigens, primarily for solubility and recovery of target molecules. Inclusion of affinity tags may have unintended consequences on a protein's solubility and susceptibility to aggregation, as well as altering immunogenicity. The usefulness of inclusion of protein tags may be outweighed by their impact on the proteins structure and function, stressing the need for careful clone design considerations early in product development.

11:15 Expression of Therapeutic Full-Length Monoclonal Antibodies in Pichia pastoris

Leon F. Garcia-Martinez, Ph.D., Director, Target Therapeutics, Alder Biopharmaceuticals

We have developed a rapid, robust technology platform for production of full-length humanized monoclonal antibodies in Pichia pastoris. This expression system captures all the attributes provided by this host (low cost, strain stability, etc.) while eliminating product development risk associated with untested expression systems. These Pichia produced antibodies display identical biochemical and pharmacokinetic characteristics of conventionally produced antibody proteins. The platform has been successfully run under GMP at 2000L scale with excellent reproducibility. Our first molecule, ADL518 has already been successfully used in human clinical trials.

11:45 Bio-Layer Interferometry as a Tool in the Bioprocess Analytical Laboratory

Sarah Koob, Scientist, Analytical Research and Development, Pfizer
Bio-Layer Interferometry (BLI) with the ForteBio Octet QK has proved to be a valuable tool in our bioprocess development analytical laboratory.   For our platform MAbs, as well other proA binding protein drug candidates, protein A Octet titer assays are employed  for primary and secondary screening of cell lines,  scale-up culture and cell line stability monitoring,  and bioreactor titer performance monitoring.  Assay results, turnaround time and labor are compared with HPLC titer assay as well as ECL immunoassay titer assay.  For protein drug candidates that do not bind protein A, several custom biosensor titer assays were developed using the StreptAvidin Biosensor as a platform. 

12:15 Genome to Proteome: Developing Expression Clone Resources

Susan Celniker, Ph.D., Head, Department of Genome Dynamics; Co-Director Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory

We describe a method for high-throughput production of protein expression-ready clones and two clone sets for expression, one in tissue and another in live animals. We have made over 11,000 clones ranging in size from 78 bp to 6699 bp with a median of 1056 bp. The donor clone facilitates rapid and simple transfer of the ORF into any expression vector of choice. Vectors are available for expressing these ORFs in bacteria, cell lines, or transgenic animals. The flexibility of this ORF clone collection makes possible a variety of proteomic applications, including protein interaction mapping, high throughput cell based expression screens, and functional studies. We have transferred 5800 ORFs to a vector that allows production of a FLAG-HA tagged protein in  tissue culture cells with a methallothionein inducible promoter.  These clones are being used to produce a protein complex map of Drosophila.

12:45 Lunch on Your Own

2:00 BuzZ Session A

2:45 Refreshment Break in the Exhibit Hall with Poster Awards

3:30 BuzZ Session B

4:15 End of Conference

4:15 Registration for Short Courses

4:30 – 7:30 Concurrent Dinner Short Courses (SC5-SC9)*

*Separate Registration Required.

Day 1 | Day 2 | Download Brochure

Links to Companion Meetings

Pipeline 4

Choosing, Designing and Optimizing Hosts and Platforms

Overcoming Challenges, Finding Solutions