Methods and compositions for the display of polypeptides on the pili of gram- positive bacteria

Patent metadata

Field	Value
Publication number	US-2016304567-A1
Application number	US-201414513797-A
Country	US
Kind code	A1
Filing date	Oct 14, 2014
Priority date	Dec 19, 2007
Publication date	Oct 20, 2016
Grant date	—

Abstract

Provided herein are methods and compositions for the display of polypeptides of interest on the tip of pili of Gram-positive bacteria. According to the present invention, the polypeptide of interest is amino terminal to a Gram-positive bacterial pilus tip protein or an active variant or fragment thereof, wherein the active variant or fragment comprises a cleaved cell wall sorting signal (CWSS) motif. The Gram-positive bacterium displaying a polypeptide of interest on the tip of pili that are disclosed herein are useful, for example, in methods for immunizing a subject with an antigen and methods for removing contaminants from a composition.

First claim

What we claim:

1 . A recombinant nucleic acid encoding a pilus tip fusion protein comprising an antigen fused to a carboxy-terminus of a fragment of Cpa comprising a cell wall sorting signal (CWSS) motif, wherein the pilus tip fusion protein comprises an amino-terminal sec-dependent signal sequence.

2 . The recombinant nucleic acid of claim 1 wherein the CWSS motif comprises SEQ ID NO: 1.

3 . The recombinant nucleic acid of claim 1 wherein the CWSS motif comprises SEQ ID NO: 10 or SEQ ID NO: 11.

4 . The recombinant nucleic acid of claim 1 wherein the fragment of Cpa comprising the CWSS motif comprises is a polypeptide having at least 50% sequence identity to SEQ ID NO: 101.

5 . The recombinant nucleic acid of claim 1 wherein the amino-terminal dec-dependent signal sequence is a polypeptide having at least 40% sequence identity to SEQ ID NO: 100.

6 . The recombinant nucleic acid of claim 1 further comprising genes sipA2, tee3, and srtC2.

7 . The recombinant nucleic acid of claim 5 wherein the sipA2 gene expresses a polypeptide having at least 40% sequence identity to SEQ ID NO: 14.

8 . The recombinant nucleic acid of claim 5 wherein the srtC2 gene expresses a polypeptide having at least 40% sequence identity to SEQ ID NO: 7.

9 . The recombinant nucleic acid of claim 5 wherein the tee3 gene expresses a polypeptide having at least 40% sequence identity to SEQ ID NO: 12.

10 . An expression cassette comprising the recombinant nucleic acid of claim 1 .

11 . A vector comprising the expression cassette or claim 10 .

12 . The expression cassette of claim 10 further comprising genes sipA2, tee3, and srtC2.

13 . The expression cassette of claim 12 wherein the sipA2 gene expresses a polypeptide having at least 40% sequence identity to SEQ ID NO: 14.

14 . The expression cassette of claim 12 wherein the srtC2 gene expresses a polypeptide having at least 40% sequence identity to SEQ ID NO: 7.

15 . The expression cassette of claim 12 , wherein the tee3 gene expresses a polypeptide having at least 40% sequence identity to SEQ ID NO: 12.

16 . A pili expressing bacteria comprising the recombinant nucleic acid of claim 1 and a nucleic acid comprising genes sipA2, tee3, and srtC2, wherein expression of the genes and the pilus tip fusion protein localizes the pilus tip fusion protein to the tip of pili.

17 . The pili expressing bacteria of claim 16 , wherein the sipA2 gene expresses a polypeptide having at least 40% sequence identity to SEQ ID NO: 14.

18 . The pili expressing bacteria of claim 16 , wherein the srtC2 gene expresses a polypeptide having at least 40% sequence identity to SEQ ID NO: 7.

19 . The pili expressing bacteria of claim 16 , wherein the tee3 gene expresses a polypeptide having at least 40% sequence identity to SEQ ID NO: 12.

Assignees

• Univ Emory

Inventors

• Scott June
• Zähner Dorothea
• Quigley Bernard

Classifications

• A61P37/04

  Immunostimulants · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K2319/035

  containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins · CPC title

• C12Y304/22

  Cysteine endopeptidases (3.4.22) · CPC title