Methods for producing soluble, biologically-active disulfide-bond containing eukaryotic proteins in bacterial cells
AssigneeBoard of Regents, The University of Texas System
InventorsGeorge Georgiou, Marc Ostermeier
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Abstract
Disclosed are methods of producing eukaryotic disulfide bond-containing polypeptides in bacterial hosts, and compositions resulting therefrom. Co-expression of a eukaryotic foldase and a disulfide bond-containing polypeptide in a bacterial host cell is demonstrated. In particular embodiments, the methods have been used to produce mammalian pancreatic trypsin inhibitor and tissue plasminogen activator (tPA) in soluble, biologically-active forms, which are isolatable from the bacterial periplasm. Also disclosed are expression systems, recombinant vectors, and transformed host cells.
Claims40
A process for producing in a bacterial cell, a biologically-active, soluble eukaryotic polypeptide having at least about three disulfide bonds, comprising expressing in said cell a first DNA segment encoding a disulfide isomerase operably linked to a signal sequence and a second DNA segment encoding said eukaryotic polypcptide operably linked to a signal sequence under conditions effective to produce said eukaryotic polypeptide.
An expression system for producing in a bacterial cell, a biologically-active, soluble eukaryotic polypeptide, said system comprising a first DNA segment and a second DNA segment, wherein said first segment encodes a disulfide isomerase and said second segment encodes a eukaryotic polypeptide having at least about three disulfide bonds.
A recombinant vector comprising a first transcriptional unit encoding a mammalian protein disulfide isomerase operably linked to a first signal sequence and said second transcriptional unit comprises a DNA segment encoding a mammalian polypeptide having at least about three disulfide bonds operably linked to a second signal sequence.
Dependencies are inferred from the claim text.