title: Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation.
abstract: Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK; Biocenter Oulu, University of Oulu, P.O. Box 5400, FIN-90014 Finland; Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 5400, FIN-90014 Finland.
authors.forename: Heli I
authors.initials: HI
authors.lastname: Alanen
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Walker
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Bernard Katz Building, London WC1H 0AH, UK.
authors.forename: M
authors.initials: M
authors.lastname: Lourdes Velez Suberbie
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
authors.forename: Cristina F R O
authors.initials: CF
authors.lastname: Matos
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Bönisch
authors.affiliation: School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Freedman
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Bernard Katz Building, London WC1H 0AH, UK.
authors.forename: Eli
authors.initials: E
authors.lastname: Keshavarz-Moore
authors.affiliation: Biocenter Oulu, University of Oulu, P.O. Box 5400, FIN-90014 Finland; Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 5400, FIN-90014 Finland.
authors.forename: Lloyd W
authors.initials: LW
authors.lastname: Ruddock
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK. Electronic address: c.robinson-504@kent.ac.uk.
authors.forename: Colin
authors.initials: C
authors.lastname: Robinson
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000502:physiology*; D019382:Human Growth Hormone / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000502:physiology*; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified; D010084:Oxidation-Reduction; D019897:Periplasm / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000906:Antibodies; D004220:Disulfides; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C408585:twin-arginine translocase complex, E coli; D019382:Human Growth Hormone
keywords: Biopharmaceuticals; Cell engineering; E. coli; Protein export; Tat pathway