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Digital Course: E. coli Innovations


CONFERENCE SERIES: Bioprocess & Manufacturing 

Recorded at: The Bioprocessing Summit 


Order DVD2012 SC4 DVD Cover About this Product: 

Escherichia coli has proven its worth as a protein expression platform. Currently, E.coli is not viewed so much as an ‘alternative’ platform, but as a viable choice for achieving high-level expression of human genes and proteins at a reasonable cost. This digital course provides an extensive and indepth overview to expressing proteins using E.coli and also explores strategies for successful E.coli protein expression, including: 


  • Host cell engineering to improve product quality
  • Development of protein expression assays
  • Development and optimization of protein purification processes
  • Automating high-throughput protein expression and purification
  • Scaling up production

This digital course is perfect for those who want to learn about using E.coli as an expression system, and for researchers wishing to build and expand their mastery.

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About this Product:
3 Presentations
121 Slides
Over 161 Minutes
Individual: $345
Site License: $1380

Agenda At A Glance: 

Overview—Protein Expression with Escherichia coli

Cynthia KinslandCynthia L. Kinsland, Ph.D., Research Associate, Biotechnology Center, and Head, Protein Production Facility, Cornell University

Most proteins are not present in high enough levels to be economically purified in useful quantities from their natural hosts; therefore, recombinant expression systems are frequently employed. E. coli has several advantages over other systems: genetic manipulations are straightforward, the cultures are facile to grow, media is inexpensive and many recombinant proteins express to high levels in soluble and active form. Despite its limitations, E. coli has proven to be capable of producing a wide range of classes of proteins. Possible causes of failure to express well in E. coli include toxicity of the target protein, codon bias problems, poor protein folding, proteolysis of the expressed protein and mRNA problems (instability and hairpin formation). Despite the predicted high failure rate, the low cost and convenience of E. coli make it a rational starting point for attempting to obtain a recombinant protein. This talk will provide an overview of the methods available, and will outline a reasonable starting strategy as well as options and rescue strategies for the more troublesome projects.

Biography: Dr. Kinsland obtained a B.S. in chemistry and a B. A. in French literature from the University of Southwestern Louisiana and an M. S. and Ph. D. in bio-organic chemistry from Cornell University.  Since 2001 she has directed the Protein Production and Characterization core facility at Cornell University.   She focuses on the production of proteins for crystallography.

Advances in Recombinant Protein Expression and Purification

Francis RajamohanFrancis Rajamohan, Ph.D., Senior Principal Scientist, Exploratory Medicinal Sciences, Pfizer Inc.

The increasing demand for milligram quantities of highly quality recombinant proteins for drug discovery and protein therapeutics warrants the needs for improved and efficient expression and purification technologies. Recombinant proteins are often produced in expression systems such as E. coli, insect cells, yeast, mammalian cells, and cell-free systems. Among these methods, the E. coli expression system is still considered the workhorse because of the ease, speed and cost. In addition, E. coli generated reagents are more homogeneous in terms of lack of post translational modifications which could promote the crystallizability of proteins. In this presentation, some of the advances in the growing field of protein expression and purification of recombinant proteins will be discussed.

Biography: Senior Principal Scientist (2001 to Present), Exploratory Medicinal Sciences, Central Research Division, Pfizer Inc. CT: Responsibilities include: (1) construct design/refinement, cloning and expression in E. coli, insect cells and mammalian cells; (2) Scale-up production using wave-bag bioreactors; (3) affinity purification, and biochemical characterization of proteins to support exploratory drug discovery, high throughput screening, structure-based drug design, and biomarker development; (4) generation of stable and transient mammalian cell lines; (5) technology and assay development (BacMam technology/ BIIC (Baculo Infected Insect Cells) technology); (6) collaborate with other technology areas (X-ray/NMR/biophysics/safety) and therapeutic areas (7) drive projects from idea to drug design.

Director (1997 to 2001), Department of Protein Engineering, Parker Hughes Cancer Center, MN: Headed the protein engineering group with 12 associates whose responsibility extended from expression construct design, protein expression, purification, characterization, and assay development through production of protein crystals. Developed and applied cutting edge technologies (Gene shuffling, alanine scanning mutagenesis, rRNA extraction, in vitro transcription/translation) necessary to elucidate the mechanism of antiviral activity of ribosome inactivating proteins (RIP’s). Performed functional characterization of proteins using biophysical tool such as surface plasmon resonance (BIA-CORE). Isolated inclusion bodies, solubilized and refolded into biologically active form. Also, generated several single-chain antibodies using phage display system.

Post Doctoral Research Associate (1992-1997) Department of Biochemistry, The Ohio State University, OH: Extensively used site-directed mutagenesis techniques to study the mechanism of action of Bacillus thuringiensis (BT) larvicidal toxins to pave the way for the construction of more potent and broader range second-generation toxins.

Teaching E. coli New Tricks: ESETEC - A Family of Secretory E. Coli Strains Validated for cGMP Manufacturing of Recombinant Proteins

Guido SeidelGuido Seidel, Ph.D., Head, Process Development, Deputy Managing Director, Wacker Biotech GmbH

The talk will focus and describe the use of Wacker’s E. coli-based secretion technology (Brand name: ESETEC®) for the expression of novel classes of therapeutic proteins, the so called next generation biopharmaceuticals (e.g. Fabs). Selected case studies will be presented.

Wacker’s secretory system enables

  • expression of correctly folded proteins directly to the culture medium
  • high fermentation titers
  • easy recovery of these proteins from the culture medium
  • increased process efficiency and overall yield
  • reduced cost of goods by lowering the amount of DSP steps


Biography:
Guido Seidel, Ph.D., born in 1969 and originally trained as chemist. Specialized during PhD studies in large scale upstream technologies and biochemistry by working on 60.000L scale fermentation optimization.
 

Joined sanofi aventis Germany in 1998, formerly Hoechst AG, as fermentation specialist and was promoted to several Head-of-positions in biopharmaceutical process development and production dealing with recombinant proteins from microbial and mammalian expression systems. Moved to Wacker mid of 2007 to be responsible as deputy managing director for the operational departments at Wacker Biotech. Has a strong focus on overall and integrated bioprocess development and production from small too large scale. 

About the Conference: 

Each year, Boston hosts CHI’s "The Bioprocessing Summit" which focuses on the most pressing challenges of bioproduction. From cell line selection and transfection, to biopreservation, scale-up and bioreactor design, this event explores the bioprocesses that continually consume time and resources. The six meetings comprising the Summit provide a close-up look at the current strategies and innovations for optimizing bioprocesses while mitigating risk and ensuring bottom-line quality.

Rather than ‘mile-high’ perspectives, the Summit experts examine the steps and protocols they have refined to help you with your work at the bench, and reveal their engineering and process breakthroughs that move bioprocessing into the future. The event features interactive small-group discussions, exhibits, panel discussions, extensive poster display, and networking with colleagues from around the world.



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