Monomeric red fluorescent proteins

• US 7,671,185 B2
• Filed: 05/15/2007
• Issued: 03/02/2010
• Est. Priority Date: 04/07/2004
• Status: Active Grant

• Current Assignee: University of Chicago, University of Washington
• Original Assignee: University of Chicago, University of Washington
• Inventors: Scalley-Kim, Michelle, Baker, David, Glick, Benjamin S., Strongin, Daniel E., Bevis, Brooke
• Primary Examiner(s): Desai; Anand U
• Assistant Examiner(s): LIU, SAMUEL W
• Application Number:
  US11/748,641
• Publication Number:
  US 20070244298A1
• Time in Patent Office: 1,022 Days
• Field of Search: None
• US Class Current: 536/23.1
• CPC Class Codes: C07K 14/43504 from invertebrates
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: Y10S 530/802 Chromogenic or luminescent peptides

Abstract

Disclosed are nucleotide sequences encoding monomeric variants of DsRed fluorescent proteins and methods of use thereof.

Claims

1. An isolated polynucleotide encoding a variant polypeptide of wild-type DsRed of SEQ ID NO:

2, the variant polypeptide having reduced oligomerization relative to wild-type DsRed, the variant polypeptide comprising the amino acid substitutions K83M, K163H, and Y193H, the variant polypeptide further comprising at least one amino acid substitution selected from the group consisting of E26Y, K92T, V96S, T106E, T108Q, I125K, S131A, I180V, and M182K relative to SEQ ID NO;

2.

2. The isolated polynucleotide of claim 1, wherein the polypeptide comprises the amino acid substitution I125K or I180V.

3. The isolated polynucleotide of claim 1, wherein the polypeptide comprises at least one amino acid substitution selected from the group consisting of R149K, R153Q, H162S, L174T, E176D, Y192N, R216H, H222S, L223G, and F224S.

4. The isolated polynucleotide of claim 1, wherein the variant polypeptide further comprises at least one amino acid substitution selected from the group consisting of V71A, C117T, V175C, S179T, S203N, and G219A.

5. The isolated polynucleotide of claim 1, wherein the variant polypeptide further comprises at least one amino acid substitution selected from the group consisting of R2D, S3N, and S4T.

6. The polynucleotide of claim 1, wherein the variant polypeptide further comprises R2A, K5E, N6D, T21S, H41T, N42Q, V44A, A145P, and T217A.

7. An isolated polynucleotide encoding a variant polypeptide of wild-type DsRed of SEQ ID NO:

2, the variant polypeptide having reduced oligomerization relative to wild-type DsRed, wherein the variant matures more rapidly than wild-type DsRed, the variant polypeptide comprising;

at least one amino acid substitution selected from the group consisting of K83M, K83L, K163Q, K163M, K163H, and Y193H;

at least three amino acid substitutions selected from the group consisting of E26Y, K92T, V96S, T106L, T108Q, I125K, S131A, I180V, and M182K;

at least three amino acid substitutions selected from the group consisting of R149K, R153Q, H162S, L174T, L176D, Y192N, R216H, H222S, L223G, and F224S; and

at least one amino acid substitution selected from the group consisting of V71A, C117T, V175C, S179T, S203N, and G219A relative to SEQ ID NO;

2.

8. The polynucleotide of claim 7, wherein the variant polypeptide further comprises R2A, K5E, N6D, T21S, H41T, N42Q, V44A, A145P, and T217A.