Compositions related to controllable intervening protein sequences (CIPS) comprising reversible zinc-binding motifs and inteins

Patent metadata

Field	Value
Publication number	US-9796967-B2
Application number	US-201314418858-A
Country	US
Kind code	B2
Filing date	Aug 1, 2013
Priority date	Aug 1, 2012
Publication date	Oct 24, 2017
Grant date	Oct 24, 2017

Abstract

Disclosed are compositions comprising an engineered intein designed such that the self-cleaving activity of the intein can be modulated by a zinc-binding motif as well as methods and systems for making and using the compositions.

First claim

What is claimed is:

1. A modified peptide comprising a controllable intervening protein sequence (CIPS), wherein the modified peptide comprises the structure:
X 1 -CIPS
wherein X 1 is an affinity tag, wherein the CIPS comprises the amino acid sequence of a reversible zinc-binding motif and an intein, wherein the reversible zinc-binding motif is appended to the N-terminus of the intein, and wherein the reversible zinc-binding motif comprises G-E-G-H (SEQ ID NO: 1) or G-D-G-H (SEQ ID NO: 2).

2. The modified peptide of claim 1 , wherein the modified peptide comprises the structure
X 1 -CIPS-X 2
wherein X 2 comprises a protein of interest.

3. The modified peptide of claim 1 , wherein the reversible zinc-binding motif comprises the sequence G-E-G-H-H (SEQ ID NO: 3).

4. The modified peptide of claim 1 , wherein the reversible zinc-binding motif comprises the sequence G-E-G-H-G (SEQ ID NO: 4).

5. The modified peptide of claim 1 , wherein the reversible zinc-binding motif comprises the sequence G-D-G-H-H (SEQ ID NO: 5).

6. The modified peptide of claim 1 , wherein the reversible zinc-binding motif comprises the sequence G-D-G-H-G (SEQ ID NO: 6).

7. The modified peptide of claim 1 , wherein the CIPS comprises SEQ ID NO: 11, 15, 19, 23, 27, or 31.

8. The modified peptide of claim 1 , wherein the nucleic acid encoding the CIPS comprises SEQ ID NO: 12, 16, 20, 24, 28, or 32.

9. The modified peptide of claim 1 , wherein the cleavage rate of the intein is reduced by 10% or more when compared to the modified peptide of claim 1 lacking the reversible zinc-binding domain.

10. A method for binding and eluting a phage-displayed polypeptide from a protein of interest, wherein said method comprises:
(a) producing the modified peptide of claim 2 ;
(b) binding the modified peptide of step (a) to a solid support;
(c) contacting a phage-displayed polypeptide with the support-bound modified peptide of step (b), thereby permitting binding of the phage-displayed polypeptide to the support-bound modified peptide of step (b);
(d) removing any unbound phage-displayed polypeptide; and
(e) eluting the bound phage-displayed polypeptide by inducing cleavage of the protein of interest.

11. The method of claim 10 , wherein the modified peptide is exposed to a chemical agent which inhibits splicing or cleavage of the protein of interest until step (d).

12. The method of claim 11 , wherein after step (d), the chemical agent which inhibits splicing or cleavage is removed, thereby allowing splicing or cleavage of the protein of interest.

13. The method of claim 11 , wherein the chemical agent is zinc.

Assignees

• Univ Ohio State
• Ohio State Innovation Foundation

Inventors

• Ma Buyong
• Nellis David F
• Zhu Jianwei S
• Wood David W

Classifications

• C12N9/16 Primary

  acting on ester bonds (3.1) · CPC title

• C07K14/4702

  Regulators; Modulating activity · CPC title

• C12P21/02

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

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K2319/20

  containing a tag with affinity for a non-protein ligand · CPC title