Cleavage resistant photoluminescent proteins and applications thereof

• US 10,442,842 B2
• Filed: 03/02/2017
• Issued: 10/15/2019
• Est. Priority Date: 03/02/2016
• Status: Active Grant

• Current Assignee: The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Original Assignee: The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Inventors: Beitle, Robert R., Mukherjee, Rudra Palash, Thallapuranam, Suresh K., Sakon, Joshua, McNabb, David S.
• Primary Examiner(s): Gebreyesus, Kagnew H
• Application Number:
  US15/448,282
• Publication Number:
  US 20170253640A1
• Time in Patent Office: 957 Days
• CPC Class Codes: B01D 15/08 Selective adsorption, e.g. chromatography
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae

Abstract

Photoluminescent proteins resistant to protein cleavage reagents are described herein. In being resistant to cleavage reagents, such photoluminesncent proteins can reduce the number of digestion or cleavage products, thereby simplifying quantification and purification of recombinant protein compositions. In some embodiments, a photoluminescent protein described herein comprises an amino acid sequence having an absence of internal methionine residues.

Claims

1. A photoluminescent protein comprising:

an amino acid sequence having an absence of internal methionine residues, wherein each internal methionine residue is substituted with an amino acid inert to cyanogen bromide (CNBr), and at least three internal methionine residues are each substituted by a leucine residue.

2. The photoluminescent protein of claim 1, wherein the photoluminescent protein is a green fluorescent protein.

3. The photoluminescent protein of claim 2, wherein the amino acid sequence of the green fluorescent protein includes the sequence of SEQ ID NO:

1.

4. The photoluminescent protein of claim 1, wherein the photoluminescent protein is selected from the group consisting of blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein and red fluorescent protein and wherein the internal methionine residues are substituted with one or more amino acids inert to CNBr.

7. The recombinant protein composition of claim 1, wherein the amino acid sequence of the green fluorescent protein includes the sequence of SEQ ID NO:

1.

16. The photoluminescent protein of claim 1, wherein the photoluminescent protein substantially retains its native photoluminescent intensity.

17. The photoluminescent protein of claim 2, wherein the green fluorescent protein is a GPF_UV variant.

18. The photoluminescent protein of claim 1, wherein the photoluminescent protein is substantially free of additional amino acid mutations.

5. A recombinant protein composition comprising:

a first amino acid sequence of a target protein; and

a photoluminescent protein coupled to the first amino acid sequence, the photoluminescent protein comprising an amino acid sequence having an absence of internal methionine residues, wherein each internal methionine residue is substituted with an amino acid inert to CNBr and at least three of the substituted amino acids is leucine.

6. The recombinant protein composition of claim 5, wherein the photoluminescent protein is a green fluorescent protein.

8. The recombinant protein composition of claim 5, wherein the photoluminescent protein is selected from the group consisting of blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein and red fluorescent protein and wherein the internal methionine residues are substituted with one or more amino acids inert to CNBr.

13. The method of claim 8, wherein the photoluminescent protein is selected from the group consisting of blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein and red fluorescent protein and wherein the internal methionine residues are substituted with one or more amino acids inert to CNBr.

14. The method of claim 8 further comprising separating the recombinant protein from the intact photoluminescent protein.

15. The method of claim 14, wherein separating comprises loading the recombinant protein and intact photoluminescent protein on a chromatographic column.

9. A method of purifying recombinant protein composition comprising:

providing a mixture including the recombinant protein composition, the recombinant protein composition comprising a first amino acid sequence of a target protein and a photoluminescent protein coupled to the first amino acid sequence, the photoluminescent protein including an amino acid sequence having an absence of internal methionine residues; and

treating the recombinant protein composition with CNBr to release the recombinant protein from an intact photoluminescent protein.

10. The method of claim 9, wherein the photoluminescent protein is a green fluorescent protein and wherein the internal methionine residues are substituted with one or more amino acids inert to CNBr.

11. The method of claim 10, wherein the one or more substitute amino acids is leucine.

12. The method of claim 11, wherein the amino acid sequence of the green fluorescent protein includes the sequence of SEQ ID NO:

1.

19. A photoluminescent protein comprising:

an amino acid sequence having an absence of internal methionine residues, wherein internal methionine residues are substituted with amino acids inert to cyanogen bromide (CNBr), and the photoluminescent protein retains photoluminescent intensity of an unsubstituted photoluminescent protein comprising the internal methionine residues.