Reversible regulation of intein activity through engineered new zinc binding domain

Patent metadata

Field	Value
Publication number	US-10323235-B2
Application number	US-201715707588-A
Country	US
Kind code	B2
Filing date	Sep 18, 2017
Priority date	Aug 1, 2012
Publication date	Jun 18, 2019
Grant date	Jun 18, 2019

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. An isolated nucleic acid encoding 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, and
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 zinc-binding motif comprises G-E-G-H (SEQ ID NO: 1) or G-D-G-H (SEQ ID NO: 2).

2. The isolated nucleic acid encoding 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 isolated nucleic acid encoding 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 isolated nucleic acid encoding 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 isolated nucleic acid encoding 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 isolated nucleic acid encoding 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. A vector comprising the nucleic acid of claim 1 .

8. A cell line comprising the vector of claim 7 .

9. A mammalian cell line comprising the isolated nucleic acid of claim 1 .

10. A method of producing a modified peptide comprising a CIPS, the method comprising:
(a) preparing the nucleic acid of claim 1 ;
(b) transforming a host cell with the nucleic acid; and
(c) culturing the transformed host cell under conditions suitable for the expression of the modified peptide comprising a CIPS encoded by the nucleic acid.

11. The method of claim 10 , further comprising isolating the modified peptide comprising a CIPS.

12. The method of claim 10 , wherein the modified peptide comprises a protein of interest.

13. The method of claim 12 , wherein the protein of interest is a therapeutic protein.

14. The method of claim 10 , wherein the host cell is a mammalian cell.

15. The method of claim 14 , wherein the mammalian cell is a CHO cell.

16. The method of claim 10 , comprising the additional step of:
(d) exposing the modified peptide comprising the CIPS to a chemical reagent which inhibits splicing or cleavage.

17. The method of claim 16 , wherein the chemical reagent is zinc.

18. The method of claim 17 , wherein the zinc is at a concentration of 1000 μM or less.

19. The method of claim 17 , wherein the zinc is at a concentration of greater than 200 μM.

20. The method claim 17 , wherein the zinc is at a concentration of 600 μM.

21. The method of claim 17 , further comprising, after step (d):
(e) exposing the modified peptide to a chelating agent, a change in pH, a change in temperature, dialysis, or dilution, thereby removing zinc.

Assignees

• Ohio State Innovation Foundation

Inventors

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

Classifications

• C07K2319/92

  containing an intein ("protein splicing")domain · CPC title

• C07K1/22

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

• C12P21/02

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

• C07K14/4702

  Regulators; Modulating activity · CPC title

• C12N9/16 Primary

  acting on ester bonds (3.1) · CPC title