A novel heterologous overexpression system for mammalian membrane protein expression, purification and crystallization
Innovative Products and Technologies
Technology Offer
A novel heterologous overexpression system for mammalian membrane protein expression, purification and crystallization
- RAMOT at Tel Aviv University Ltd.
- From Israel
- Responsive
- Innovative Products and Technologies
Summary of the technology
Cloning and expression methods as well as constructs were developed, that yields significantly higher expression of heterogeneous membrane proteins in bacterial cells, hence facilitating proteins research and crystallization abilities. This is achieved by addition of hydrophilic alpha-helical segments to the desired membrane protein, via a cloning procedure. Two different helices enable construction of eight derivatives of the desired protein. The helices facilitate crystallization by providing the necessary hydrophilic surfaces for crystal formation.
Eight mammalian membrane proteins and two bacterial protein were overexpressed in our bacterial system.
All fusion proteins were solubilized by relatively mild detergents.
All fusion proteins were present in distinct size - monomers or dimers.
All of them were purified standard purification techniques.
The yields of the purified proteins were between 3.6-6.5mg (mammalian) to 3-10mg (bacterial) per liter culture.
All tested proteins were amenable to be concentrated and detergent exchanged for crystallization.
Tested proteins demonstrated both in vitro and in vivo activity
Project ID : 2-2007-22
Details of the Technology Offer
The Technology
A bacterial cloning and expression system for heterogeneous membrane protein expression (mammalian, bacterial) has been developed. It yields significantly higher expression of heterogeneous membrane proteins in bacterial cells, hence facilitating proteins research and crystallization abilities. This is achieved by addition of hydrophilic alpha-helical segments to the desired membrane protein, via a cloning procedure. Two different helices (hydrophilic bacterial proteins (YaiN (α) and YbeL(β)), enable construction of eight derivatives of the desired protein. The helices facilitate crystallization by providing the necessary hydrophilic surfaces for crystal formation.
Data-to date
10 membrane proteins have been purified (yields: 3.6-6.5mg (mammalian) to 3-10mg (bacterial) per liter culture) including divalent metal ion transporter from E. Coli and mammalian neurotransmitter transporters
The Need
Membranes are the site for the regulation of numerous biological processes and activities; control of these processes is mostly performed by membrane bound proteins. Expression and crystallization of membrane proteins is, however, a formidable task. Over-expression in bacteria encounters problems due to overloading of the membrane and the crystallization process is hampered by the lack of hydrophilic regions crucial for forming inter-molecular bonds in the crystal.
The absence of detailed structural information on membrane proteins severely limits drug discovery efforts and confounds elucidation of numerous mechanisms fundamental to biology. Currently the Pharma industry has to rely on molecular modeling of structures which is often unreliable, hence the great need for reliable structural data that is best derived from crystal structures.
Patent status
Granted patents US, DE, FR, UK