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A novel heterologous overexpression system for mammalian membrane protein expression, purification and crystallization (Ramot)

Summary

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 Information

Granted patents US, DE, FR, UK

ID No.

2-2007-22

Country/Region

Israel