Refolding of membrane proteins

Abstract

In a method for production of membrane receptors folded into their native structure, first, receptors solubilized in a first detergent are provided. To induce folding of receptors into their native form, the first detergent is exchanged for a second detergent. Both for the first and for the second detergent, examples are shown.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of co-pending U.S. application Ser. No. 10 / 069,433 filed on Feb. 19, 2002, which is a continuation of International application PCT / EP00 / 07763 filed on Aug. 10, 2002, and claims priority of German patent application DE 199 39 246.3 filed on Aug. 19, 1999, all of which are incorporated herein by reference in their entireties.FIELD OF THE INVENTION [0002] The present invention relates to a method for the production of proteins folded into their native or active structure, said protein being from the group of membrane receptors, excluding G-protein-coupled receptors, preferably ion channels, comprising: [0003] providing a protein from the group of membrane receptors solubilized in a first detergent, and [0004] exchanging said first detergent for a second detergent, to induce the folding of said protein into its native or active form. DESCRIPTION OF THE RELATED ART [0005] For membrane proteins, suc...

AI Technical Summary

Benefits of technology

[0021] In view of the above, it is an object of the present invention to improve the method mentioned above to reach—with good reproducibility—a high yield of the protein in active or native structure.

[0034] In this connection, it is particularly preferred if alkyl-phosphorylcholine with a chain length of C10-C16 is used as said second detergent. The inventors have recognized that this detergent provides for a high yield of refolded protein.

[0038] The expression of the DNA sequence coding for the protein according to the invention as fusion protein has, in comparison with the direct expression without carrier protein, the advantage that the carrier protein protects the protein which is desired, but, however, unknown to the expression system, against degradation by proteases and may result in a higher expression level. In particular by using glutathione-S-transferase (GST) as carrier protein, the solubility of proteins being expressed in large quantities is increased in the host cell and isolation is facilitated. The carrier protein can, further, be used for purifying fusion proteins, if suitable antibodies are provided. The same applies for purification methods with affinity chromatography.

[0043] In this method, it is advantageous that, in comparison with the use of pure detergent micelles, the yield of native protein can even be improved. The lipid extract of the tissue, in which the receptor occurs naturally, can also be simulated by using lipids with a similar composition or by mixing same.

Problems solved by technology

Both publications are based on the problem that membrane proteins, to which receptors also belong, can be produced in large quantities with the help of expression vectors in bacteria, but that the protein produced, however, is not active.

For this purpose, on the one hand, in eukaryotic cells (cells of mammals or insects), functional protein can be produced, however, the systems are expensive, and the expression rates are low, which is also disadvantageous.

In view of the above, the two publications mentioned at the outset describe methods, in which the protein is expressed in the inner part of the cell, where it, however, aggregates, and hence is not functionally available.

Although the methods for the production of membrane protein or receptor protein that are described in the publications mentioned above lead to active structures, the methods described, according to the knowledge of the inventors of the present application here submitted, are insofar not satisfying, as the yield is low and the method is poorly reproducible.

Images