Red fluorescent proteins with enhanced bacterial expression, increased brightness and reduced aggregation

• US 8,679,749 B2
• Filed: 11/03/2008
• Issued: 03/25/2014
• Est. Priority Date: 11/01/2007
• Status: Active Grant

• Current Assignee: University of Chicago
• Original Assignee: University of Chicago
• Inventors: Glick, Benjamin S., Keenan, Robert, Strongin, Daniel E., Strack, Rita L., Bhattacharyya, Dibyendu
• Primary Examiner(s): PROUTY, REBECCA E
• Application Number:
  US12/740,019
• Publication Number:
  US 20110020784A1
• Time in Patent Office: 1,968 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae

Abstract

Polynucleotides encoding variant polypeptides of DsRed are provided herein. The DsRed variants have increased bacterial expression, reduced aggregation, increased solubility, shifted emission spectra or increased brightness relative to a wild-type DsRed.

Claims

1. An isolated polynucleotide encoding a variant polypeptide of SEQ ID NO:

1, the variant polypeptide having fluorescence, having reduced aggregation relative to SEQ ID NO;

1, having a sequence identity of 90% or more with SEQ ID NO;

1 or SEQ ID NO;

5, and comprising a proline residue at position 10 of SEQ ID NO;

1 or SEQ ID NO;

5.

2. The polynucleotide of claim 1, wherein the variant polypeptide comprises an aspartate residue at position 2 of SEQ ID NO:

1 or SEQ ID NO;

5 and a threonine residue at position 4 of SEQ ID NO;

1 or SEQ ID NO;

5.

3. The polynucleotide of claim 1, wherein the variant polypeptide comprises at least one amino acid selected from the group consisting of a methionine residue at position 66, a threonine or alanine residue at position 73, a lysine, cysteine, or arginine residue at position 75, a cysteine residue at position 175 and an alanine residue at position 219, relative to SEQ ID NO:

1 or SEQ ID NO;

5.

4. The polynucleotide of claim 1, wherein the variant polypeptide further comprises at least one amino acid selected from the group consisting of a lysine residue at position 36, a glutamine residue at position 47, a histidine residue at position 121, a leucine residue at position 141, a glycine residue at position 169, a valine residue at position 210, and a glutamine residue at position 225, relative to SEQ ID NO:

1 or SEQ ID NO;

5.

5. The polynucleotide of claim 1, wherein the variant polypeptide further comprises at least one amino acid selected from the group consisting of a threonine residue at position 117, a proline residue at position 145, and an alanine residue at position 217, relative to SEQ ID NO:

1 or SEQ ID NO;

5.

6. The isolated polynucleotide of claim 1, wherein the polynucleotide further encodes a polypeptide of interest linked to the variant polypeptide, the polypeptide of interest and the variant polypeptide being expressed as a fusion protein.

7. A cell comprising the sequence of claim 1.

8. A construct comprising the polynucleotide of claim 1 operably connected to a promoter.

9. A vector comprising the construct of claim 8.

10. A method of obtaining expression of a variant polypeptide of SEQ ID NO:

1 comprising introducing the vector of claim 9 into a host cell under conditions that permit expression of the variant polypeptide.

11. The method of claim 10, wherein the polynucleotide further encodes a polypeptide of interest linked to the variant polypeptide, the polypeptide of interest and the variant polypeptide being expressed as a fusion protein.

12. The method of claim 10, further comprising evaluating the expression of the variant polypeptide by detecting red fluorescence.

13. The method of claim 12, further comprising monitoring temporal or spatial changes in red fluorescence.

14. The polynucleotide of claim 4, wherein the variant polypeptide comprises a glycine residue at position 169, relative to SEQ ID NO:

1 or SEQ ID NO;

5.

15. The polynucleotide of claim 1, wherein the variant polypeptide comprises a lysine residue at position 188, relative to SEQ ID NO:

1 or SEQ ID NO;

5.

16. The polynucleotide of claim 15, wherein the variant polypeptide further comprises a glycine residue at position 169, relative to SEQ ID NO:

1 or SEQ ID NO;

5.

17. The isolated polynucleotide of claim 16, wherein the polynucleotide further encodes a polypeptide of interest linked to the variant polypeptide, the polypeptide of interest and the variant polypeptide being expressed as a fusion protein.

18. A cell comprising the polynucleotide of claim 17.

19. A construct comprising the polynucleotide of claim 17 operably connected to a promoter.

20. A vector comprising the construct of claim 19.

21. A method of obtaining expression of a variant polypeptide of SEQ ID NO:

1 comprising introducing the vector of claim 19 into a host cell under conditions that permit expression of the variant polypeptide.