Membrane Protein Expression Using Nanobody Stabilization
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Solution Overview
Problem
Current methods face challenges in achieving high expression levels and conformational stability of membrane proteins, particularly GPCRs, due to their low natural expression, hydrophobic nature, and biochemical instability, which hampers structural and functional characterization and drug discovery efforts.
Innovation Solution
The use of yeast cells co-expressing membrane proteins with Nanobodies directed against them, allowing for increased expression, direct purification, and stabilization of membrane proteins in specific conformational states, facilitating structural and functional studies and drug screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If membrane proteins are overexpressed in recombinant systems, then expression levels increase, but conformational stability and functional integrity deteriorate
Solution Approach 1:
The patent uses solubility-enhancing fusion partners (such as GST, MBP, or TRX) as intermediary molecules that are fused to the membrane protein of interest. These fusion partners act as mediators that improve the solubility and conformational stability of the hydrophobic membrane protein during overexpression, while allowing the membrane protein to maintain its functional integrity. The fusion partner can be subsequently removed to obtain the native membrane protein.
Solution Approach 2:
The patent employs chemical chaperones (such as glycerol, sugars, or osmolytes) and biochemical modifications to alter the expression and purification parameters. By changing the chemical environment and physical conditions during expression and purification, the patent maintains membrane protein stability at higher expression levels without compromising conformational integrity.
2Reliability
If membrane proteins are purified from native tissue, then structural integrity is maintained, but expression levels remain too low for sufficient material
Solution Approach 1:
The patent employs affinity tags (such as His-tags, FLAG-tags, or GST-tags) that are self-assembled on the membrane protein during expression. These tags enable the protein to be purified directly from the expressing cells through affinity chromatography, eliminating the need for complex multi-step purification procedures from native tissue while maintaining structural integrity and achieving high yields.
3Reliability
If membrane proteins are stabilized by fusion with bacteriophage T4 lysozyme, then structural stability improves, but the protein may not represent the true natural conformation
Solution Approach 1:
The patent uses removable affinity tags and solubility-enhancing fusion partners that can be easily removed after purification. Unlike permanent fusion with T4 lysozyme, these temporary auxiliaries serve their purpose during expression and purification but can be cleaved off to yield the native membrane protein in its authentic conformation, thus providing both stability during production and authenticity in the final product.
Data Source
AI summary
The disclosure relates cells or cellular systems that express both a membrane protein and a binding domain directed to the membrane protein. Also, methods are provided that use such cells or cellular systems to produce higher amounts of the membrane proteins. Further, the cells or cellular systems can be used as tools for the structural and functional characterization of membrane proteins, as well as for screening and drug discovery efforts targeting membrane proteins.