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Overview | Short
Courses | Day 1 |
Day 2 | Day 3 |
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TUESDAY, JANUARY 13
7:00am – 6:00pm Registration Open
7:30 Breakfast Workshop
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Sponsored by |
Slonomics® – A Unique Technology for the Generation of Highly Designed Gene Libraries
Thomas Waldmann, Ph.D., Science and Technology Applications, Sloning BioTechnology
Sloning specializes in the synthesis of highly genetically diverse and precise
customized gene libraries for protein engineering up to 1011 different variants. Unlike traditional methods that rely on single-stranded oligonucleotides, the patented Slonomics® technology uses a set of double-stranded DNA triplets as universal building blocks. Predefined triplets represent all possible sequence combinations required to synthesize any gene – ‘one codon at a
time.’ For library production, multiple codons can be introduced in parallel during the synthesis of a gene construct at any desired sequence position. The absence of functional bias and the ability to select and precisely control delivery up to 20 specific codons per sequence position and at any ratio results in exceptionally high quality libraries containing the complete set of desired mutants. In this talk, new technology advancements regarding the random integration of codons into gene sequences and practical examples for improved screening success with SlonoMax™ mutant libraries will be presented.
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OVERCOMING EXPRESSION DIFFICULTIES
8:15 Chairperson’s Remarks
John E. Harlan, Ph.D., Abbott Laboratories
8:20 Removal of C-terminal Polyhistidine Tags and/or Unstructured Residues from Recombinant Proteins with a Genetically Engineered Fungal A-type Carboxypeptidase
David S. Waugh, Ph.D., Senior Investigator, Macromolecular Crystallography Laboratory NCI-Frederick
The use of a genetically engineered form of carboxypeptidase A from the fungal entomopathogen Metarhizium anisopliae (MeCPA) for the removal of hexahistidine tags from the C-termini of recombinant proteins will be described, with emphasis on actual case studies. This His-tagged enzyme has been modified to prevent autodigestion and its substrate specificity has been thoroughly characterized.
8:50 How to Make Antibiotics Obsolete: Use of Bacterial Selection Modules for Efficient Protein Production in E. coli
Philippe
Gabant, CEO, Delphi Genetics
Antibiotic resistance genes are the most common selectable markers used in fermentation processes to avoid the overgrowth of plasmid free cells in the culture. Delphi Genetics has designed a novel and highly effective stabilization system, called StabyTM, based on the use of selection modules naturally found in plasmids, bacterial chromosomes and bacteriophages. This genetic stabilization technology solves the problem of plasmid instability (without requiring a specific medium) and insures that upon induction 100% of the bacteria will produce the recombinant protein leading to higher yields of the target protein and less background caused by unwanted proteins.
9:20 Functional Protein Analysis in DNA-Based Coupled Transcription/Translation Cell-free Systems
Kate Zhao, Ph.D., Sr. Research Scientist, Proteomics, Promega Corporation
Using four DNA based cell-free systems prepared from different organisms (S30 extract from E. coli, insect cell extract, wheat germ extract and rabbit reticulocyte lysate), protein expression and function can be quickly analyzed. Functional protein kinase expression and protein-protein interactions can be evaluated in these systems, especially when combined with HaloTag® fusion technology. Therefore, a complete platform of DNA based coupled transcription/translational systems prepared from different organisms can greatly simply the process of protein expression and functional screening.
9:50 Coffee Break in the Exhibit Hall
10:45 Purification and Characterization of Recombinant Human Renin for X-ray Crystallization Studies
Zhongren Wu, Ph.D., Discovery Biology, Vitae Pharmaceuticals, Inc.
Renin is a member of the aspartic acid protease family and plays a key role in the renin-angiotensin system (RAS) that regulates blood pressure and extracellular volume in the body. In our structure-based drug design efforts, we have developed a novel procedure, in which concanavalin A is used, to purify full-length recombinant renin from mammalian cell cultures, instead of inconvenient affinity columns. After activation with trypsin to remove propeptide, fully active renin is used successfully in biological assays and co-crystallization with inhibitors for X-ray studies.
11:15 Overexpression, Solubilization, and Purification of Human Glucagon-Like Peptide Type 1 Receptor
Mark Chiu, Ph.D., Department of Structural Biology, Abbott Labs
We present strategies and technologies used to over-express, solubilize, and purify human type 1 Glucagon-like Peptide receptor, a type II G protein-coupled receptor. We outline how such technology to prepare milligram quantities of GLP-1R can be applied to other human membrane proteins.
11:45 Expression, Purification and Characterization of the Human OATP2B1 Membrane Transporter from the Sf9 Insect Cell System
Bill Tschantz, Ph.D., Lab Head – Biologics Center, Protein Production and Antibodies, Novartis Institutes for BioMedical Research
OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. This work was undertaken to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we show the successful expression, purification and characterization of OATP2B1 in a heterologous expression system which is being used to develop in vitro and in silico model systems for xenobiotic disposition.
| 12:15pm Comparative Study on Autologous Expression Improvement in Human Cells
by Gene Optimization: Results and Applications |
Sponsored by |
Stephan Fath, Ph.D., Scientist Research & Development,
Geneart AG
In silico expression
optimization in conjunction with rapid in vitro gene synthesis is a
state-of-the-art technique to circumvent problems even in
autologous expression. We
report the largest gene expression study on synthetic optimized genes in
mammalian cells to date. 50 human genes from the NCBI Entrez
database representing
different protein classes such as protein kinases, cytokines,
membrane proteins and transcription factors, were optimized for
increased mRNA halflife and
protein expression in human cells. The
optimized sequences and their wildtype counterparts were
synthesized de novo and cloned into expression
vectors. Expressed protein levels in HEK‑293T
cells were quantified and compared.
The results clearly indicate a significant improvement of
expression yield with
optimized constructs compared to respective wildtype versions.
Therefore, gene synthesis is not only a versatile manner to obtain
individualized genes but also allows for autologous expression
increase in most cases. |
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| 12:30 Luncheon Workshop
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Sponsored by
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Tools for Heterologous Protein Expression in the Yeast Kluyveromyces lactis
Christopher Taron, Ph.D., Research Division Head, Gene Expression, New England Biolabs, Inc.
The yeast Kluyveromyces lactis is an efficient host for heterologous protein expression that has been used to produce proteins at industrial scale for nearly two decades. New England Biolabs offers a kit for K. lactis expression and a growing set of host strains and methods that address common protein expression problems. This talk will provide a technical overview of NEB’s K. lactis toolbox. |
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ENHANCING PROTEIN EXPRESSION
2:00 Chairperson’s Remarks
Dominic Esposito, Ph.D., Principal Scientist, Group Leader, Clone Optimization Group, SAIC-Frederick, Inc.
2:05 Tuning Escherichia coli for Membrane Protein Over-Expression
Jan-Willem de Gier, Ph.D., Associate Professor, Center for Biomembrane Research
at Stockholm University, Sweden, Xbrane bioscience AB
A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have engineered an E. coli BL21(DE3) derived strain, Lemo21(DE3), that is tunable for over-expression. Our strain conveniently allows optimizing overexpression of both membrane and also soluble proteins using only a single strain protein rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
2:35
Nanolipoprotein
Particles (NLPs) as Scaffolds for Generation of Membrane Protein Complexes
using Cell-Free Technologies
Matthew
A. Coleman, Ph.D., Senior Scientist, Biosciences and Biotechnology
Division, Physics and Life Sciences, Lawrence Livermore National
Laboratory
We
have developed cell-free methods for producing soluble membrane proteins
contained in nanolipoprotein particles (NLPs). NLPs have shown great
promise as a biotechnology platform for solubilizing and characterizing
membrane proteins. However, current approaches are limited because they
require extensive efforts to express, purify and solubilize the membrane
protein prior to insertion into NLPs. By the simple addition of a few
constituents to cell-free extracts, we can produce soluble membrane
proteins in NLPs with considerably less effort. Importantly, the approach
also provides a single-step process for the production of functional
soluble membrane proteins that eliminates the need for cell growth, cell
lysis, and subsequent purification, refolding etc. We have demonstrated
this process on multiple membrane proteins important for proton
translocation, host-pathogen interactions and cell surface signaling. Our
technique represent a unique solution to solubility and purification
problems associated with membrane proteins.
3:05 Biochemical Characterization of Different Acid
Alpha-glucosidases for Treatment of Pompe Disease
Karen L. Lee, Director, Structural Protein Chemistry, Genzyme Corporation
| 3:35
Optimization of Gene Design for Heterologous Expression |
Sponsored by |
Mark Welch, Ph.D., Senior Scientist, DNA 2.0
Optimization of heterologous expression systems is critical for efficient production of high value protein products and can be essential for generating sufficient material for structure based drug design and functional studies. While much work has been done in expression vector and host strain development, surprisingly little has until now been accomplished in the area of gene sequence optimization. With support from NSF and multiple collaborations across the biotechnology industry, DNA2.0 has actively studied the relationship of gene design to heterologous expression yield. Using its gene synthesis capacity, DNA2.0 has created large systematically varied gene sets for multiple gene types and analyzed expression in several commonly utilized host systems. Striking differences are observed for the dependence of expression on synonymous codon usage in different hosts. In E.
coli, protein expression levels varied over two orders of magnitude for each of two gene sets tested. Our data show that codon bias is a strong determinant of expression level in E.
coli; however, the preferred codon bias is distinctly different from that of genes naturally highly expressed in the bacterium and no correlation is seen between expression and the codon adaptiveness index. With collaborators we have also synthesized and tested systematically varied gene sets for multiple target proteins in multiple expression hosts. The correlation between codon bias and protein expression in multiple systems is compared. |
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3:50 Refreshment Break in the Exhibit Hall
4:30 BuzZ Sessions
Moderated Small Group Breakout Discussions
Time has been designated for Interactive Roundtable Discussions with a specific topic assigned to each group. This unique opportunity will bring together both conference attendees to discuss challenges, solutions and future collaborations.
Have a topic idea? Want to host a table?
We welcome all attendees to suggest a topic(s) that would be of interest.
Please send all suggestions to Mary Ann Brown at mabrown@healthtech.com
Click
here for a listing of BuzZ Session topics
5:30 Close of Day
Overview | Short
Courses | Day 1 |
Day 2 | Day 3 |
Download Brochure
For more information, please contact:
Elizabeth Lamb, Conference Producer
Phone: 207-493-7874
E-mail: elamb@healthtech.com
For exhibit and sponsorship information, please contact:
Suzanne Carroll, Manager-Business
Development
Phone: 781-972-5452
E-mail: scarroll@healthtech.com
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