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Publication Number
US-9322011-B2
Publication Date
2016-04-26
Expiration Date
Abstract
There is provided a fusion protein suitable for secretion of more than one polypeptide(s) of interest (POI) comprising a signal peptide, a POI, a passenger domain comprising a beta stem domain from an autotransporter protein, and a translocator domain from an autotransporter protein, wherein the beta stem-forming sequence of the passenger domain is essentially intact and the POI(s) is/are fused to the beta stem domain.
Core Innovation
The invention relates to improved Gram negative bacterial host cells that express more than one POI using a fusion protein or a nucleic acid encoding the fusion protein. The fusion protein includes a passenger domain with a beta stem domain from an autotransporter protein belonging to the pfam autotransporter family PF03797, where the beta stem forming sequence is essentially intact, and a translocator domain also deriving from an autotransporter protein belonging to PF03797.
A key feature is that at least two POI:s are inserted into, replace, or partly replace separate side domains of the passenger domain. The passenger domain in native form comprises at least two side domains, and a signal peptide targets the fusion protein to the inner membrane of Gram negative bacteria.
The described system is usable for cell surface display and soluble secretion and can further include cleavage between the passenger and the translocator. The documented embodiments include outer membrane vesicles and bacterial ghosts, and they emphasize maintaining correct processing and enabling secretion and/or surface display using autotransporter scaffolds such as SPATEs including Hbp and EspC.
Claims Coverage
The provided claim set includes three independent claims that cover a Gram negative bacterial host cell, a fusion protein, and a nucleic acid arranged for expression of that fusion protein. Across these independent claims, the core inventive architecture is the PF03797 passenger beta stem and translocator combined with a signal peptide for inner-membrane targeting, together with multiple POI insertion, replacement, or partial replacement into at least two distinct passenger side domains.
Gram negative bacterial host cell with PF03797 passenger beta stem and multi-POI side-domain replacement
A Gram negative bacterial host cell capable of expressing more than one POI, wherein the host cell comprises a fusion protein or a nucleic acid encoding a fusion protein having a passenger domain comprising a beta stem domain from an autotransporter protein belonging to PF03797 with an essentially intact beta stem forming sequence, a translocator domain from an autotransporter protein belonging to PF03797, and a signal peptide able to target the fusion protein to the inner membrane of Gram negative bacteria; wherein the passenger domain in native form comprises at least two side domains and at least two POI:s are inserted into, replace or partly replace separate side domains.
Fusion protein with PF03797 passenger beta stem and multi-POI side-domain replacement
A fusion protein comprising more than one POI, a passenger domain comprising a beta stem domain from an autotransporter protein belonging to PF03797 with an essentially intact beta stem forming sequence, a translocator domain from an autotransporter protein belonging to PF03797, wherein the passenger domain in native form comprises at least two side domains and at least two POI:s replace or partly replace a separate side domain.
Nucleic acid for expression of PF03797 multi-POI passenger/translocator fusion with intact beta stem and in-frame cloning sites
A nucleic acid arranged for expression of a fusion protein, comprising in frame a sequence encoding a signal peptide able to target the fusion protein to the inner membrane of Gram negative bacteria, a sequence encoding a passenger domain comprising a beta stem domain from an autotransporter protein belonging to PF03797, and a sequence encoding a translocator domain deriving from an autotransporter protein belonging to PF03797; wherein the sequence encoding the passenger domain in native form comprises at least two stretches encoding side domains protruding from the beta stem domain, wherein the passenger domain sequence comprises at least two stretches of cloning site sequence that allow in-frame cloning of at least two DNA sequences encoding POI:s, said cloning site sequences being inserted into, replacing or partly replacing separate stretches of the side-domain stretches, and the encoded beta stem forming protein sequence is essentially intact.
Across the independent claims, the inventive coverage centers on a PF03797-based autotransporter architecture with an essentially intact passenger beta stem and a PF03797 translocator, combined with a signal peptide for inner-membrane targeting and enabling expression of multiple POI:s by inserting, replacing, or partly replacing at least two passenger side domains, with corresponding nucleic-acid designs that preserve the beta stem while providing in-frame cloning sites for multiple POI sequences.
Stated Advantages
Enables a Gram negative bacterial host cell to express more than one POI using a single fusion protein architecture.
Supports cell surface display and/or soluble secretion via autotransporter-based fusion design using a PF03797 passenger beta stem with an essentially intact beta-stem forming sequence.
Enables multi-antigen presentation by inserting or replacing multiple POI:s in separate passenger side domains.
Supports specific display formats such as outer membrane vesicles and bacterial ghosts.
Documented Applications
Cell-surface display of fusion proteins on Gram-negative bacterial hosts, including display from outer membrane vesicles and bacterial ghosts.
Soluble secretion and/or secretion-associated display of antigen POI:s from engineered autotransporter fusion constructs.
Presentation of Mycobacterium tuberculosis antigens as POI:s, including ESAT-6, Ag85B, TB10.3, TB10.4, and Rv2660c.
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