2017 Archived Content
THURSDAY, 16 NOVEMBER
PROTEIN PURIFICATION STRATEGIES:
DEALING WITH PROTEINS THAT ARE PRONE TO AGGREGATE
This course will provide a comprehensive and detailed outline of hands-on issues for purifying proteins. We will first address general considerations about the protein we want to produce, including issues of activity, solubility, homogeneity, purity,
and proper oligomeric conformation. Aggregation is one of the main obstacles in protein production, so we will look at how to monitor for aggregation and comprehend its mechanism. We will also discuss how to check for the optimal solubility conditions
at the expression level, and our comprehensive approach for optimizing solubility during purification. We will also discuss expression screening methodology, environmental factors to consider during purification, families of additives, and screening
for additives. Lastly, we will address ways to avoid aggregation, as well as setting up protein concentration and storage.
Mario Lebendiker, Ph.D., Head, Protein Purification Facility, Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem
- General considerations about the protein we want to produce.
- Activity, solubility, homogeneity, purity, proper oligomeric conformation and other considerations.
- Aggregation: one of the main obstacles in protein production. Mechanism of aggregation. When does aggregation start? How do we know that we are dealing with a prone to aggregate protein?
- What to do at the expression level.
- Domain design
- Fusion proteins
- Change expression conditions
- Change expression system
- Optimize purification procedure
- How to check optimal solubility conditions at the expression level
- Expressing screening methodology for prone-to-aggregate proteins. HTP screening, or minimized hierarchical screening platform
- Environment factors to consider during purification steps: cell lysis, purification , concentration and storage
- pH , buffering system and ionic strength
- Moderate chaotrope as protein-protein interaction reducers
- Kosmotropic environment as protein stabilizer
- Surfactants and non-denaturative detergents
- Reducing agents
- Families of additives
- Methods for monitoring protein aggregates. Different approaches for screening solubility conditions. Advantages and disadvantages
- Our screening methodology to select the best additives. Examples
- Other key issues to avoid aggregation during processing:
- Purification time
- Protein concentration in each step
- Prevention of mechanical or not mechanical stresses
- Our comprehensive approach for optimizing solubility during purification
- Set-up protein concentration and storage
Instructor Bio Sketch:
Mario Lebendiker, Ph.D., Head, Protein Purification Facility, Wolfson Centre for Applied Structural Biology, The
Hebrew University of Jerusalem
Dr. Mario Lebendiker is in charge of the Protein Purification Facilities at the Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem. He is actively involved in many collaborations for structural and biochemical studies within
the Hebrew University, other Universities in Israel, as well as with biotech and pharmaceutical companies. Dr. Lebendiker received a PhD in Biochemistry in 1982 from the Animal Virology Center (CEVAN), in Buenos Aires University, Argentina. Together
with many other laboratories, he found the Protein Production and Purification Partnership in Europe (P4EU) network; a platform for the exchange of information, know-how and materials between core facility labs in the field of protein expression and