Harvard College

The University of Texas System

Other

Jonathan R Beckwith

George Georgiou

Daniel Ritz

Paul Henry Bessette

Fredrik Aslund

Jackie Eun-ah Lim

Charles S Hoffman

Andrew Lewis Wright

Dana Boyd

Feras Hatahet

Michael Syvanen

Melinda Faulkner

Kathryn L Strauch

Colin Manoil

Jessica Blazyk

Stephanie Gon

Ralph R Isberg

Provided herein are host cells or host cellular expression systems that express a membrane protein. Also, methods are provided that use such host cells or host cellular expression systems to produce higher amounts of the membrane proteins. Further, the cells or cellular systems can be used as tools for the functional characterization of membrane proteins, as well as for screening and drug discovery efforts targeting membrane proteins.

Host cells and systems for membrane protein expression

The invention provides composition and methods for producing proteins of interest which comprise at least one disulfide bond, include proteins which in their mature form do not contain disulfide bonds, but whose precursor molecule contained at least one disulfide bond. The methods employ a host cell modified to more efficiently produce properly folded disulfide bond containing proteins. The host cells generally contain a mutation in one or more reductase genes, and can be further genetically modified to increase their growth rate, and are further optionally modified to increase the expression of a catalyst of disulfide bond formation. Host cells, methods for u sing such to produce proteins of interest, proteins of interest produced by these methods are within the scope of the invention.

Prokaryotic host cells for expressing proteins rich in disulfide bonds

The invention provides composition and methods for producing proteins of interest which comprise at least one disulfide bond, include proteins which in their mature form do not contain disulfide bonds, but whose precursor molecule contained at least one disulfide bond. The methods employ a host cell modified to more efficiently produce properly folded disulfide bond containing proteins. The host cells generally contain a mutation in one or more reductase genes, and can be further genetically modified to increase their growth rate, and are further optionally modified to increase the expression of a catalyst of disulfide bond formation. Host cells, methods for using such to produce proteins of interest, proteins of interest produced by these methods are within the scope of the invention.

Compositions and methods for production of disulfide bond containing proteins in host cells

This invention features a method of isolating a mutant strain of Escherichia coli, having a defective periplasmic protease, the method comprising the steps of: mutagenizing an E. coli cell, wherein the cell comprises: (a) an inner and an outer membrane, (b) a periplasmic space between the membranes, (c) a protein which in a first state is mobile, being able to move through the outer membrane and enter medium surrounding the cells, the protein in the first state being detectable in the medium, and in a second state is not mobile, remaining inside the cell, and (d) a periplasmic protease which converts the protein from the second state to the first state in the cell, and selecting a mutant cell which produces a reduced level of the detectable protein in the medium compared to the E. coli cell, wherein the mutant cell comprises the defective periplasmic protease. This invention also features mutant strains of E. coli having a defective periplasmic protease.

Periplasmic protease mutants of Escherichia coli

An export sequence of export DNA can be identified by transforming a population of cells with a vector having a transposon that includes a structural gene encoding a detectable compound, positioned between insertion sequences. The structural gene encodes a detectable compound that, in wild-type organisms, is translated with an export peptide effective to export the compound. Since the transposon lacks DNA coding for an export sequence capable of exporting the detectable gene product, transformants that export the gene product include a DNA fusion of the transposon to export DNA from the parent cell, in a position to allow expression of the fused DNA. The transformants are analyzed to locate the export DNA or a gene comprising it, and the position of the export DNA in the cell's genome is determined; the orientation of the insertion also is determined. Having identified cell genes that naturally contain export DNA, the export DNA is cloned and fused to a gene encoding a product whose production and export are desired. For example, a cell is engineered to produce and export a desired product, to enable easier substance recovery and to improve cell tolerance for high levels of the desired product.

Export of intra-cellular substances

Growing community of inventors

Cambridge, MA, United States of America

Jonathan R Beckwith