PepTalk 2017
PepTalk 2017

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Cambridge Healthtech Institute’s 2nd Annual
Membrane Proteins
A Valuable Resource and Target
January 19-20, 2016

Membrane proteins are the gateways to the cell and are valuable drug targets. For researchers to design better-targeted drugs, they need to know their structure and functional characteristics. Cambridge Healthtech Institute’s 2nd Annual Membrane Proteins conference addresses the strategies and solutions for their extraction, expression and purification, and features case studies showcasing their value as a drug target. Join the in-depth exploration of how to obtain functional membrane proteins, and learn more about this important protein class.

Final Agenda

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1:00 pm Conference Registration


2:00 Chairperson’s Opening Remarks

William Gillette, Ph.D., Senior Scientist, Protein Expression Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research (FNL)


2:05 Making Water-Soluble Integral Membrane Proteins in vivo Using an Amphipathic Protein Fusion Strategy

Matthew_DeLisaMatthew P. DeLisa, Ph.D., William L. Lewis Professor, Chemical & Biomolecular Engineering, Cornell University

Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic ‘shield’ that sequesters the IMP from water and promotes its solubilization.

⊲ Featured Presentation
2:45 Expression and Sample Preparation of Membrane Proteins for Structure Determination by NMR

Stanley_OpellaStanley Opella, Ph.D., Professor, Chemistry and Biochemistry, University of California, San Diego

The advantages of heterologous expression of proteins in bacteria include the ability to make relatively large amounts and the ready incorporation of stable isotopes. The use of a hydrophobic fusion protein enables the sequestration in inclusion bodies to avoid damaging the cell membrane.

3:15 Refreshment Break in the Exhibit Hall with Poster Awards

Fujifilm Diosynth4:00 Overcoming the Purification Challenges with Bone Morphogenetic Proteins

Robertson_PatrickPatrick Robertson, Ph.D., Senior Scientist, Purification Development, FUJIFILM Diosynth Biotechnologies

The distinctive nature of bone morphogenetic proteins creates unique challenges in purification, such as low solubility near physiological pH and a tendency to aggregate and/or precipitate in the presence of salts limiting the design space for developing an effective and scalable purification strategy. We will present an approach that leverages their unique structural characteristics in purification development.

4:30 It Takes Two to Tango—Structure/Function Studies Yield the Dance of the Permease

Ron_KabackH. Ronald Kaback, M.D., Distinguished Professor, Physiology, University of California, Los Angeles

Lactose permease (LacY) catalyzes translocation of a galactoside and an H+ across the membrane. X-ray structures, and structure/function studies reveal that: (1) LacY utilizes an alternating access mechanism; (2) sugar binding involves induced fit; (3) Active transport does not involve a change in K<sub>D</sub> for sugar on either side of the membrane, but the pK<sub>a</sub> decreases markedly. (4) Transport is driven chemiosmotically, and ∆µ̃H+ acts kinetically to accelerate the process.

5:00 Strategies for High Yield Affinity Purification of Functional G Protein Coupled Receptor from Detergent Solutions

Alexei_YeliseevAlexei Yeliseev, Ph.D., Staff Scientist, LMBB, NIH/NIAAA

Human cannabinoid receptor CB2, a G protein-coupled receptor involved in regulation of immune response, is an important target for pharmaceutical drug development. We expressed the functional CB2 receptor in E. coli, and optimized its purification by tandem affinity chromatography using novel affinity resins StrepTactin XT Superflow and EF2 Ca-calbindin-based resin. Examples of successful purification and efficient recovery (over 80%) of CB2 from dilute detergent-containing solutions will be presented.

5:30-5:45 Short Course Registration

5:45-8:45 Dinner Short Courses

Recommended Short Course: SC10: Transient Protein Production in Mammalian Cells

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8:00 am Conference Registration and Morning Coffee


8:30 Chairperson’s Remarks

Henry C. Chiou, Ph.D., Associate Director, Cell Biology, Life Science Solutions, Thermo Fisher Scientific

8:35 Light-Controlled Intracellular Delivery of Native Peptides and Proteins

Norbert_ReichNorbert O. Reich, Ph.D., Professor, Chemistry & Biochemistry, University of California, Santa Barbara

We have developed a nanoparticle-based platform to deliver proteins into specific cells with spatiotemporal control achieved through the use of highly penetrating near-infrared light. The delivery of therapeutic peptides as well as transcription factors provides a means to control the timing and amount of release for synthetic biology and translational applications.

9:05 Cell-Free Expression of High-Quality GPCRs in Eukaryotic and Prokaryotic Lysates

Ralf-Bernhardt Rues, Research Fellow, Institute of Biophysical Chemistry, Goethe University Frankfurt

GPCRs are crucial regulators of cellular physiology and play central roles in medical research. The development of efficient GPCR production pipelines based on synthetic biology is discussed. Key issues will be systems adaption to individual GPCRs, folding optimization with defined nanodisc and lysate combinations and directed engineering for sample stabilization. Presented examples are human endothelin as well as catecholamine binding receptors. GPCR sample properties obtained from either bacterial or insect cell lysates will be compared.

9:35 Automated Transient Transfection for High Throughput Protein Production

Suh_ChrisChris Suh, Ph.D., Business Development, PhyNexus, Inc.

Transient transfection of mammalian cell lines is being implemented by the pharmaceutical industry to produce the therapeutic protein candidates very rapidly compared to previous technology thus allowing large numbers of drug candidates to be screened and studied. However, high throughput automated transient transfection is required to cope with the dramatically increased sample load. Here we describe the integration, implementation and validation of different robotic platforms for automated transient transfection of mammalian cells.

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

10:50 Nanodisc in Drug Discovery: Assembly, Characterization and Application

Han_XuHan Xu, Ph.D., Principal Scientist, Amgen, Inc.

Integral membrane proteins (IMPs) are of therapeutic interest and are targeted by a majority of approved drugs. It’s difficult to express, purify and maintain the functional conformation of IMPs. Nanodisc presents a reliable method to solubilize and stabilize IMPs in detergent-free condition. In this presentation, I detail assembly and characterization of KcsA-Kv1.3 Nanodisc and demonstrate applications of Nanodisc in drug discovery.

11:20 Developing a Targeted Integration CHO Host for Clinical & Commercial Cell Line Development

Yongping_CrawfordYongping Crawford, Ph.D., Scientist, Early Stage Cell Culture, Genentech, Inc., a member of the Roche Group

11:50 Expression of Membrane Recombinant Hemagglutinins, Components of Influenza Vaccine Flublok®, Using Baculovirus Expression System

Nikolai_KhramstovNikolai Khramtsov, Ph.D., Associate Director, Upstream Development, Process Development, Protein Sciences Corp.

We developed a universal process for the GMP production of influenza recombinant hemagglutinins (rHAs), components of seasonal influenza vaccine Flublok®. The GMP manufacture in BEVS (baculovirus expression vector system) begins in less than two months from the FDA announcement of vaccine composition for a new flu season (in February of each year). Our results suggest that BEVS is a highly efficient system for expression of membrane-bound biologically active HAs.

12:20 pm ExpiCHO Transient Expression System: Comparative Data, New Applications and Tips for Maximal Performance

Zmuda_JonathanJonathan Zmuda, Ph.D., Associate Director, Cell Biology, Thermo Fisher Scientific

The ability to produce transient CHO-derived proteins early on during the drug development process is highly advantageous to minimize changes in critical quality attributes observed when progressing from discovery to bioproduction. Previous CHO-based transient systems have been hampered by low levels of protein production compared to HEK293 systems, in some instances 50-100 times less than the best 293-based systems. With the introduction of the ExpiCHO transient expression system, researchers have a flexible new tool to express proteins at significantly higher levels than in 293 cells, up to 3 grams per liter, while benefitting from the relevance of CHO cells. In this session we will present data comparing the ExpiCHO and Expi293 transient expression systems, newly-developed ExpiCHO applications notes as well as tips for ensuring easy set-up and maximal performance from the ExpiCHO transient expression system.

12:50 Session Break

Aragen horizontal1:00 Luncheon Presentation: Development of an Extended Half-Life Erythropoietin for Treating Feline Anemia

Zhan_HangjunHangjun Zhan, Ph.D., Vice President and Head, Biologics Research, Kindred Bio

Due to various market pressures that are unique from human therapeutics, cost effective manufacturing of recombinant biologics is essential for veterinary applications. Here we present the development of high titer engineered feline EPO cell line for clinical trial and commercial manufacturing. In addition, product quality characterization including biophysical properties, in vitro activities and pharmacokinetics will also be discussed.

1:30 Luncheon Presentation II (Sponsorship Opportunity Available)


2:00 Chairperson’s Remarks

Caroline Colley, Ph.D., Senior Scientist, Antibody Discovery and Protein Engineering, MedImmune

2:05 Antibodies Against Difficult to Express Membrane Protein Targets

Yelena_BisharyanYelena Bisharyan, Ph.D., Director of External Alliances, Tetragenetics, Inc.


Bill_Harriman Bill Harriman, Ph.D., CSO, Crystal Bioscience

Ion channels such as Kv1.3 have been historically difficult to raise antibodies against due to sequence conservation, paucity of cell surface epitopes, and poor expression levels in heterologous systems. Tetragenetics Inc. and Crystal Bioscience are addressing these issues by combining their unique technologies for membrane protein expression in Tetrahymena thermophila, and antibody generation in chickens, to develop therapeutic antibodies against a range of ion channel targets including Kv1.3, a voltage-dependent channel produced by effector memory T-cells implicated in certain autoimmune disorders.

2:35 Engineering Ion Channels for Structural Studies and Ligand Discovery

Susmith Mukund, Senior Research Associate, Genentech, Inc.

3:05 Antibody-Mediated Blockade of Human Orai1 Inhibits T Cell Activation in vitro and in vivo

Stefan Zahn, Ph.D., Principal Scientist, Antibody Technology, Novo Nordisk A/S

Ion channels are widely expressed on cells and tightly regulate the flow of ions between the extracellular and the intracellular environment. Dysregulation has been linked to pain, epilepsy and even autoimmune inflammatory diseases. We will present our recent work on targeting T cell specific ion channels like CRAC by antibodies inhibiting T cell activation in vivo and in vitro.

3:35 Refreshment Break in the Exhibit Hall with Poster Viewing

4:30 Approaches for Tumor Selective Targeting Using Monoclonal Antibodies

Madan_KatragaddaMadan Katragadda, Ph.D., Senior Principal Scientist, Pfizer, Inc.

Potent novel means of therapeutic intervention utilizing immune cell retargeting and antibody-drug conjugates necessitates tumor selective targets owing to their extremely high potent nature. Several strategies have evolved in the past decade to selectively target tumors by either exploiting antigens, antigen complexes and glycoepitopes that are selectively overexpressed on tumor cells relative to the normal cells or simultaneously targeting two or more antigens using antibody engineering techniques. Examples describing these strategies are presented.

5:00 Challenges in Generating Antibodies to Integral Membrane Proteins

Ramu_SadhukhanRamkrishna (Ramu) Sadhukhan, Senior Group Leader, AbbVie Bioresearch Center Center

Developing therapeutic antibodies against integral membrane proteins is difficult, as GPCRs and ion channels are often expressed at low levels on cell surface and are unstable when purified. Poor quality membrane protein immunogens has led to limited success in generating antibodies that bind native cell surface molecules and remains a bottleneck for membrane protein target validation and monoclonal antibody-based therapeutics. Here, we present antigen preparation and antibody generation against multispanner proteins.

5:45 BuzZ Session B

Join your peers and colleagues for interactive roundtable discussions.

6:30-7:30 Reception in the Exhibit Hall with Poster Viewing

7:30 Close of Conference

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