System useful for reporting protein-protein interactions in the bacterial periplasm

Patent metadata

Field	Value
Publication number	US-9388406-B2
Application number	US-200913129527-A
Country	US
Kind code	B2
Filing date	Nov 16, 2009
Priority date	Nov 17, 2008
Publication date	Jul 12, 2016
Grant date	Jul 12, 2016

Abstract

One aspect of the present invention relates to a reporter system for detection of protein-protein interactions in the periplasm of a prokaryotic host cell. The reporter system includes a first expression system which has a nucleic acid molecule encoding a first fragment of a reporter protein molecule, a nucleic acid molecule encoding a first signal sequence, and a nucleic acid molecule encoding a first member of a putative binding pair, where the nucleic acid molecule encoding the first fragment, the nucleic acid molecule encoding the first signal sequence, and the nucleic acid molecule encoding the first member of the putative binding pair are operatively coupled to permit their expression in a prokaryotic host cell as a first fusion protein. The reporter system also includes a second expression system which has a nucleic acid molecule encoding a second fragment of the reporter protein molecule, a nucleic acid molecule encoding a second signal sequence, and a nucleic acid molecule encoding a second member of the putative binding pair, where the nucleic acid molecule encoding the second fragment, the nucleic acid molecule encoding the second signal sequence, and the nucleic acid molecule encoding the second member of the putative binding pair are operatively coupled to permit their expression in a prokaryotic host cell as a second fusion protein, where, when expressed in a prokaryotic host cell, at least one of the first and the second fusion proteins are co-translationally transported to the periplasm where, when present, the first and second members of the putative binding pair bind together and the first and second fragments of the reporter protein molecule are reconstituted, thereby producing an active reporter protein. The reporter system may be used to carry out methods of identifying candidate compounds which bind to a target protein, identifying a candidate gene which modulates binding between a first protein and second protein, and identifying a candidate compound which modulates binding between a first protein and second protein.

First claim

What is claimed:

1. A reporter system for detection of protein-protein interactions in the periplasm of a prokaryotic host cell, said system comprising:
a first expression system comprising:
a nucleic acid molecule encoding a first fragment of a reporter protein molecule;
a nucleic acid molecule encoding a first signal sequence directed to a signal recognition particle (SRP) transport pathway; and
a nucleic acid molecule encoding a first member of a putative binding pair, wherein said nucleic acid molecule encoding the first fragment, said nucleic acid molecule encoding the first signal sequence, and said nucleic acid molecule encoding the first member of the putative binding pair are operatively coupled to permit their expression in a prokaryotic host cell as a first fusion protein and a second expression system comprising:
a nucleic acid molecule encoding a second fragment of the reporter protein molecule;
a nucleic acid molecule encoding a second signal sequence; and
a nucleic acid molecule encoding a second member of the putative binding pair, wherein said nucleic acid molecule encoding the second fragment, said nucleic acid molecule encoding the second signal sequence, and said nucleic acid molecule encoding the second member of the putative binding pair are operatively coupled to permit their expression in a prokaryotic host cell as a second fusion protein, wherein the first signal sequence and optionally the second signal sequence are capable of directing co-translational transport of at least one of the fusion proteins, when expressed in a prokaryotic host cell, into the host cell periplasm where, when present, the first and second members of the putative binding pair bind together and the first and second fragments of the reporter protein molecule are reconstituted, thereby producing an active reporter protein.

2. The reporter system according to claim 1 , wherein both the first signal sequence and the second signal sequence are capable of directing co-translational transport of the first and the second fusion proteins to the periplasm.

3. The reporter system according to claim 1 , wherein the first member of the putative binding pair and the second member of the putative binding pair have a binding affinity which is stronger than the binding affinity of the first fragment and the second of the reporter protein.

4. The reporter system according to claim 1 , wherein said reporter protein molecule is selected from the group consisting of a monomeric protein, a multimeric protein, a monomeric receptor, a multimeric receptor, a multimeric biomolecular complex, adenylate cyclase, alkaline phosphatase, β-lactamase, cellulase, chloramphenicol acetyl transferase (CAT), disulfide bond oxidase A (DsbA), maltose binding protein (MBP), methyltransferase, dihydrofolate reductase (DHFR), luciferase, thymidylate synthase, thymidine kinase, Trp1 N-(5′-phosphoribosyl)-anthranilate isomerase, ubiquitin, and fluorescent proteins.

5. The reporter system according to claim 4 , wherein the reporter protein molecule is β-lactamase.

6. The reporter protein molecule according to claim 5 , wherein the first fragment and the second fragment of the reporter protein molecule, respectively, comprise β-lactamase's α fragment having the amino acid sequence of SEQ ID NO: 2, and β lactamase's ω fragment having the amino acid sequence of SEQ ID NO: 3.

7. The reporter system according to claim 1 , wherein the second signal sequence is directed to a transport pathway selected from the group consisting of Sec pathway, SRP pathway, and Tat pathway.

8. The reporter system according to claim 7 , wherein the first signal sequence and the second signal sequences are directed to the SRP pathway and are selected from the group consisting ssArtI, ssDsbA, ssFlgA, ssLivJ, ssSfmC, ssSTII, ssTolB, ssTorT, ssYraP, and ssYraI.

9. The reporter system according to claim 7 , wherein the second signal sequence is directed to the Sec pathway and is selected from the group consisting ssAppA, ssBla, ssClyA, ssLep, ssMalE, ssOmpA, ssOmpT, ssOmpX, ssPelB ( Erwinia chrysanthemi ), ssPhoA, ssRbsB, and ssYebF.

10. The reporter system according to claim 7 , wherein the second signal sequence is directed to the Tat pathway and is selected from the group consisting ssFdnG, ssFdoG, ssNapG, ssNrfC, ssHyaA, ssYnfE, ssWcaM, ssTorA, ssNapA, ssYagT, ssYcbK, ssDmsA, ssYdhX, ssYahJ, ssYedY, ssCueO, ssSufI, ssYcdB, ssTorZ, ssHybA, ssYnfF, ssHybO, ssAmiC, ssAmiA, ssYfhG, ssMdoD, ssFhuD, ssYaeI, and ssYcdO.

11. The reporter system according to claim 1 , wherein the prokaryotic host cell is a gram negative bacteria.

12. The reporter system according to claim 1 , wherein the putative binding pair is selected from the group of proteins consisting a membrane protein-soluble binding protein pair, a membrane protein-membrane protein binding pair, a biotin-avidin binding pair, ligand-receptor binding pair, and an antibody-antigen pair.

13. The reporter system according to claim 12 , wherein the putative binding pair is an antibody-antigen pair, wherein the antibody is selected from the group consisting of monoclonal antibody, bispecific antibody, single-chain antibody (scAb), single-chain Fv fragment (scFv), scFv 2 , dsFv, scFv-Fc, Fab, F(ab′) 2 , F(ab′) 3 , V L , diabody, single domain antibody, camelid antibody, triabody, tetrabody, minibody, one-armed antibody, and immunoglobulin (Ig), IgM, IgE, IgA, IgD, IgG, IgG-ΔC H 2, and wherein the antigen is selected from the group consisting of cell surface receptors, proteins regulating apoptosis, proteins that regulate progression of the cell-cycle, proteins involved in the development of tumors, transcriptional-regulatory proteins, translation regulatory proteins, proteins that affect cell interactions, cell adhesion molecules, proteins which are members of ligand-receptor pairs, proteins that participate in the folding of other proteins, SNARE protein family, and proteins involved in targeting to intracellular compartments.

14. The reporter system according to claim 12 , wherein the putative binding pair is a receptor-ligand pair, wherein the receptor is selected from the group consisting of Fc receptors (FcR), single-chain MHC, and single-chain T-cell receptor (sc-TCR).

15. The reporter system according to claim 12 , wherein the putative binding pair is a membrane protein-membrane protein binding pair or membrane protein-soluble protein binding pair, wherein the membrane protein is selected from the group consisting of monotopic membrane proteins, polytopic membrane proteins, transmembrane proteins, G protein-coupled receptors (GPCRs), ion channels, SNARE protein family, integrin adhesion receptor, and multi-drug efflux transporters.

16. A method of identifying a candidate protein which binds a target protein, said method comprising:
a) providing a first expression system comprising:
a nucleic acid molecule encoding a first fragment of a reporter protein molecule;
a nucleic acid molecule encoding first signal sequence directed to a signal recognition particle (SRP) transport pathway; and
a nucleic acid molecule encoding a target protein, wherein said nucleic acid molecule encoding the first fragment, said nucleic acid molecule encoding the first signal sequence, and said nucleic acid molecule encoding the target protein are operatively coupled to permit their expression in a prokaryotic host cell as a first fusion protein and

b) providing a second expression system comprising:
a nucleic acid molecule encoding a second fragment of the reporter protein molecule;
a nucleic acid molecule encoding a second signal sequence; and

Assignees

• Delisa Matthew P
• Mansell Thomas J
• Univ Cornell

Inventors

• Delisa Matthew P
• Mansell Thomas J

Classifications

• C07K2319/61

  containing an enzyme fusion for detection (lacZ, luciferase) · CPC title

• C12N15/1055 Primary

  Protein x Protein interaction, e.g. two hybrid selection · CPC title

• C07K2319/034

  containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title