Autotransporter system
AssigneeProkarium Limited
InventorsMarc Biarnes Carrera, Annelise Soulier
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Abstract
The present invention provides for a modified autotransporter and the use of genetically engineered microorganisms comprising said modified autotransporters in the treatment of infectious and neoplastic disease. The present invention therefore also relates to vaccine and immunotherapeutic compositions comprising said genetically engineered microorganism.
Claims35
An autotransporter construct modified to permit insertion of a heterologous polynucleotide sequence that encodes a target polypeptide for translocation across the inner and outer membrane of a Gram-negative bacterium, the autotransporter comprising i) a polynucleotide sequence that encodes a N-terminal signal sequence;
- ii) a passenger region into which said heterologous polynucleotide sequence encoding the target polypeptide is to be inserted and iii) a polynucleotide sequence encoding a translocation domain;
- wherein the passenger region comprises a synthetic polynucleotide sequence flanked by restriction enzyme recognition sequences, wherein said synthetic polynucleotide sequence comprises a polynucleotide sequence that encodes a first polypeptide tag.
A method for modifying a Gram-negative bacterial autotransporter, comprising:
- i) removing the passenger domain from the passenger region, ii) introducing a synthetic polynucleotide sequence that encodes a first polypeptide tag in the passenger region flanked by restriction enzyme recognition sequences, iii) introducing a synthetic polynucleotide sequence that encodes a second polypeptide tag within the passenger region upstream of the synthetic polynucleotide sequence that encodes the first polypeptide tag and is positioned outside of the restriction enzyme recognition sequence boundaries, iv) introducing a synthetic polynucleotide sequence that encodes a linker, said sequence being downstream of the synthetic polynucleotide sequence that encodes the first polypeptide tag and is positioned outside of the restriction enzyme recognition sequence boundaries, and v) introducing a synthetic polynucleotide sequence that encodes for a cleavage site within the passenger region downstream of the synthetic polynucleotide sequence that encodes for a linker.
Dependencies are inferred from the claim text.