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Overcoming Challenges with Solutions 


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FRIDAY, JANUARY 13

7:30 am Breakfast Presentation (Sponsorship Opportunity Available) or Morning Coffee

 

OVERCOMING MEMBRANE PROTEIN AND FINICKY PROTEIN CHALLENGES 

8:15 Chairperson's Opening Remarks

8:20 Restrained Expression: A Method to Overproduce Toxic Membrane Proteins by Exploiting Operator-Repressor Interactions

Dinesh A. Yernool, Ph.D., Assistant Professor, Department of Biological Sciences, Purdue University

Toxicity often associated with rapid overexpression results in reduced biomass along with low yields of target protein. Mitigation of toxic effects was achieved using a method we call "restrained expression," a controlled reduction in the frequency of transcription initiation by exploiting the infrequent transitions of Lac repressor to a free state from its complex with the lac-operator site within a T7lac promoter that occur in the absence of the inducer isopropyl β-D-1-thiogalactopyranoside. In addition, production of the T7 RNA polymerase that drives transcription of the target is limited using the tightly regulated arabinose promoter in Escherichia coli strain BL21-AI.

8:50 Novel Strategies for Efficient Membrane Protein Expression in E. coli

François Baneyx, Ph.D., Charles W.H. Matthaei Professor, Department of Chemical Engineering, University of Washington, Seattle

Although they play key roles in cellular function and are targeted by 60% of pharmaceutical drugs, membrane proteins are notoriously difficult to express at high levels and in a functional form. I will describe here how transcription tuning and the reprogramming of molecular chaperone pathways can be used to significantly improve the yields of this class of proteins.

9:20 Bridging the Gap: A GFP-Based Strategy for Overexpression and Purification of Membrane Proteins with Intra and Extracellular C-Termini

Jennifer M. Hsieh, Ph.D., Assistant Professor of Chemistry, Department of Physical Sciences, Santa Monica College

Expression and purification of membrane proteins (MPs) is commonly monitored using C-terminally fused green fluorescent protein (GFP), but has been restricted to MPs with intracellular C-termini (Cin) due to GFP's inability to fluoresce in the Escherichia coli periplasm. Using GlycophorinA, we developed a method to convert MPs with extracellular C-termini (Cout) to Cin providing a conduit for implementing GFP reporting.

9:50 Sponsored Presentation (Opportunity Available)

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

11:00 Development of a Cell-Free Expression Platform for the Production of Recombinant Proteoliposomes and Use in Therapeutic Approaches

Jean-Luc Lenormand, Ph.D., Team Leader, HumProTher Lab, University Joseph Fourier

Cell-free protein synthesis has emerged as one of the most efficient systems for membrane protein expression.We have successfully expressed, in mg amounts, bioactive proteoliposomes containing membrane proteins for different approaches ranging from therapeutic uses (vaccines against infectious diseases, pro-apoptotic membrane proteins for treating tumours) to the production of monoclonal antibodies or the development of biomimetic artificial membranes. Results will be presented including expression of single membrane protein or co-expression of 2 or 3 membrane protein complexes embedded into lipid bilayers.

11:30 Mammalian Production of an Isotopically Enriched Outer Domain of the HIV-1 Gp120 Glycoprotein for NMR Spectroscopy

Mallika Sastry, Ph.D., Research Scientist (c), Structural Biology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health

The bacterial expression systems currently in use to obtain isotopic enrichment cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an adenovirus vector-based mammalian expression system to produce isotopically enriched (15)N or (15)N/(13)C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation.

12:00 pm Understanding and Controlling Glycation of a Recombinant Antibody in Cell Culture

Inn H. Yuk, Ph.D., Senior Group Leader, Early Stage Cell Culture, Genentech, Inc.

Protein glycation is a form of non-enzymatic glycosylation. For a particular recombinant monoclonal antibody produced by CHO cell culture, we observed unusually high levels of glycation. We will describe our efforts to understand the factors influencing glycation in the cell culture environment, and show how we used these findings to control the glycation of this antibody.

12:30 Close of Morning Session

12:45 Luncheon Presentations (Sponsorship Opportunity Available) or Lunch on Your Own

 

CHARACTERIZATION OF SUB-VISIBLE PARTICLES 

2:00 Chairperson's Remarks

2:05 Characterization of Submicron Particle Distributions in Biologics Formulations

Flaviu Gruia, Ph.D., Scientist, Analytical Biochemistry, MedImmune

Nanoparticle Tracking Analysis performance has been tested with particle standards and several different monoclonal antibody solutions. Submicron particle populations and how they evolve over time following different sample manipulations such as dilution, filtration, fill-finish, pH have been measured and will be presented as case studies. Trends observed in the submicron particle concentrations suggest that such manipulations can trigger formation of new particles due to different mechanisms. Correlations with techniques that probe different size ranges would be included.

     Sponsored by
Enzo Life Sciences Assay Designs
2:35 Fluorescence-Based Detection of Protein and Peptide Aggregates:  From Test Tube to Tissue

Wayne F. Patton, Chief Scientific Officer, Enzo Life SciencesA novel fluorescence-based aggregation assay is described that is suitable for monitoring protein /peptide aggregation generated by thermal-, mechanical agitation- and freeze-thaw-induced stresses.  Stabilized subvisible protein particle reference standards facilitate rapid generation of a standard curve by rehydration, facilitating quantification of the aggregated material.  Advantages of monitoring aggregation using the fluorescence microplate-based assay include simple implementation, ability to quantify minute levels of aggregation, ~30 minute analysis time and robust assay performance.  The described technology has been extended to the detection of inclusion bodies containing aggregates present within cultured cells and formalin-fixed paraffin-embedded brain tissue.

3:05 Characterization of Sub-Visible Particles in Therapeutic Protein Formulations

Tobias Frommknecht, Ph.D., Formulation Scientist, pRED, F. Hoffmann-La Roche Ltd

First a small volume light obscuration (LO) method will be introduced and some limitations to the microflow digital imaging (MDI) methodologies with regards to their ability to characterize sub-visible particles (SbVP) are elucidated. Secondly the effect of fluids with specific physical properties typically observed for aqueous protein formulations – such as turbid, viscous and colored fluids – is presented with regards to counting and sizing abilities of LO and MDI methods using particle suspensions of different buoyancy and contrast. Finally, the limitations and relationships of different shape attributes and the potential of particle morphology parameters will be presented.

3:35 Measurement of Protein Unfolding/Refolding Kinetics and Structural Characterization of Hidden Intermediates by NMR Relaxation Dispersion

Derrick Meinhold, Ph.D., Research Associate, Department of Molecular Biology and Skaggs Institute of Chemical Biology, The Scripps Research Institute

Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated conformational states. NMR R2 relaxation dispersion experiments were used to investigate spontaneous unfolding and refolding events of native apomyoglobin revealing a three-state unfolding mechanism leading to a molten globule. NMR chemical shifts of the transient states highlight regions of energetic frustration that crack during unfolding.

4:05 End of Conference


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Links to Companion Meetings

Pipeline 4 

Engineering Genes, Vectors, Constructs and Clones 

Choosing, Designing and Optimizing Hosts and Platforms