US-12624071-B2 - Bright and stable red fluorescent protein

US12624071B2US 12624071 B2US12624071 B2US 12624071B2US-12624071-B2

Abstract

Improved Red Fluorescent Proteins (RFPs), nucleic acids encoding the improved RFPs, and methods of generating improved RFPs are described. The improved RFP can be used to form fusion molecule and can be used in applications for which fluorescent proteins are used.

Inventors

Hau Thi Bich NGUYEN
Geoffrey S. Waldo

Assignees

TRIAD NATIONAL SECURITY, LLC

Dates

Publication Date: 20260512
Application Date: 20230330

Claims (8)

1 . A red fluorescent protein comprising an amino acid sequence having at least 97% sequence identity to (a) SEQ ID NO: 15, wherein amino acid 17 is tyrosine, amino acid 57 is serine, amino acid 98 is isoleucine, amino acid 100 is lysine, amino acid 131 is glycine, amino acid 135 is isoleucine, amino acid 176 is tryptophan, and amino acid 203 is threonine; (b) SEQ ID NO: 17 wherein amino acid 17 is phenylalanine, amino acid 57 is serine, amino acid 98 is isoleucine, amino acid 100 is lysine, amino acid 131 is glutamate, amino acid 135 is isoleucine; amino acid 176 is tryptophan, and amino acid 203 is asparagine; or (c) SEQ ID NO: 19, wherein amino acid 3 is asparagine, amino acid 25 is tyrosine, amino acid 36 is tyrosine, amino acid 45 is arginine, amino acid 83 is phenylalanine, amino acid 114 is histidine, amino acid 115 is asparagine, amino acid 139 is arginine, amino acid 174 is histidine, amino acid is 176 is histidine, amino acid 180 is isoleucine, and amino acid 219 is alanine.
2 . The red fluorescent protein of claim 1 , wherein the red fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of: (a) SEQ ID NO: 15, wherein amino acid 17 is tyrosine, amino acid 57 is serine, amino acid 98 is isoleucine, amino acid 100 is lysine, amino acid 131 is glycine, amino acid 135 is isoleucine, amino acid 176 is tryptophan, amino acid 203 is threonine, amino acid 36 is histidine, amino acid 92 is threonine, amino acid 125 is leucine, amino acid 147 is threonine, amino acid 162 is asparagine, and amino acid 196 is aspartate; (b) SEQ ID NO: 17 wherein amino acid 17 is phenylalanine, amino acid 57 is serine, amino acid 98 is isoleucine, amino acid 100 is lysine, amino acid 131 is glutamate, amino acid 135 is isoleucine; amino acid 176 is tryptophan, amino acid 203 is asparagine, amino acid 36 is histidine, amino acid 92 is threonine, amino acid 125 is leucine, amino acid 147 is threonine, amino acid 162 is asparagine, and amino acid 196 is aspartate; or (c) SEQ ID NO: 19, wherein amino acid 3 is asparagine, amino acid 25 is tyrosine, amino acid 36 is tyrosine, amino acid 45 is arginine, amino acid 83 is phenylalanine, amino acid 114 is histidine, amino acid 115 is asparagine, amino acid 139 is arginine, amino acid 174 is histidine, amino acid is 176 is histidine, amino acid 180 is isoleucine, amino acid 219 is alanine, amino acid 92 is threonine, amino acid 125 is leucine, amino acid 147 is threonine, amino acid 162 is asparagine, and amino acid 196 is aspartate.
3 . The red fluorescent protein of claim 2 , wherein the red fluorescent protein comprises the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, and wherein said sequences possess the amino acids as in (a), (b) and (c), respectively.
4 . The red fluorescent protein of claim 2 , wherein the amino acid sequence of the red fluorescent protein consists of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, and wherein said sequences possess the amino acids as in (a), (b) and (c), respectively.
5 . The red fluorescent protein of claim 3 , wherein the red fluorescent protein further comprises a fusion protein.
6 . A nucleic acid encoding the red fluorescent protein of claim 1 .
7 . The nucleic acid of claim 6 , wherein the nucleic acid is provided in a CRISPR construct.
8 . A host cell comprising the nucleic acid of claim 6 .

Description

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT This invention was made with government support under GM098177 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING The Sequence Listing written in file S167675.000_SeqListing_ST26.xml is 22 kilobytes in size, was created Mar. 22, 2023, and is hereby incorporated by reference BACKGROUND Fluorescent proteins widely used as protein tagging agents. GFP-like proteins are an expanding family of homologous, 25-30 kDa polypeptides sharing a conserved 11 beta-strand “barrel” structure. The GFP-like protein family currently comprises well over 100 members, cloned from various Anthozoa and Hydrozoa species, and includes red, yellow and green fluorescent proteins and a variety of non-fluorescent chromoproteins. A wide variety of fluorescent protein labeling assays and kits are commercially available, encompassing a broad spectrum of spectral variants. While fluorescent proteins having various spectral and stabilities have been identified, there remained a need for fluorescent proteins with improved spectral properties or increased stability. SUMMARY Described are improved red fluorescent proteins (RFPs) and nucleic acids encoding the improved RFGs. In some embodiments, an improved RFP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to: (a) SEQ ID NO: 15, wherein amino acid at position 17 is tyrosine, amino acid at position 57 is serine, amino acid at position 98 is isoleucine, amino acid at position 100 is lysine, amino acid at position 131 is glycine, and amino acid at position 135 is isoleucine, amino acid at position 176 is tryptophan, and amino acid at position 203 is threonine; (b) SEQ ID NO: 17 wherein amino acid at position 17 is phenylalanine, amino acid at position 57 is serine, amino acid at position 98 is isoleucine, amino acid at position 100 is lysine, amino acid at position 131 is glycine, and amino acid at position 135 is isoleucine, amino acid at position 176 is tryptophan, and amino acid at position 203 is threonine; or (c) SEQ ID NO: 19, wherein amino acid at position 3 is asparagine, amino acid at position 25 is tyrosine, amino acid at position 36 is tyrosine, amino acid at position 45 is arginine, amino acid at position 83 is phenylalanine, amino acid at position 114 is histidine, amino acid at position 115 is asparagine, amino acid at position 139 is arginine, amino acid at position 174 is histidine, and amino acid is at position 176 is histidine, amino acid at position 180 is isoleucine, and amino acid at position 219 is alanine (FIG. 7). In some embodiments, an improved RFP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to: (a) SEQ ID NO: 15, wherein amino acid at position 17 is tyrosine, amino acid at position 57 is serine, amino acid at position 98 is isoleucine, amino acid at position 100 is lysine, amino acid at position 131 is glycine, and amino acid at position 135 is isoleucine, amino acid at position 176 is tryptophan, amino acid at position 203 is threonine, amino acid 36 is histidine, amino acid 92 is threonine, amino acid 125 is leucine, amino acid 147 is threonine, amino acid 162 is asparagine, and amino acid 196 is aspartate; (b) SEQ ID NO: 17 wherein amino acid at position 17 is phenylalanine, amino acid at position 57 is serine, amino acid at position 98 is isoleucine, amino acid at position 100 is lysine, amino acid at position 131 is glycine, and amino acid at position 135 is isoleucine, amino acid at position 176 is tryptophan, amino acid at position 203 is threonine, amino acid 36 is histidine, amino acid 92 is threonine, amino acid 125 is leucine, amino acid 147 is threonine, amino acid 162 is asparagine, and amino acid 196 is aspartate; or (c) SEQ ID NO: 19, wherein amino acid at position 3 is asparagine, amino acid at position 25 is tyrosine, amino acid at position 36 is tyrosine, amino acid at position 45 is arginine, amino acid at position 83 is phenylalanine, amino acid at position 114 is histidine, amino acid at position 115 is asparagine, amino acid at position 139 is arginine, amino acid at position 174 is histidine, and amino acid is at position 176 is histidine, amino acid at position 180 is isoleucine, amino acid at position 219 is alanine, amino acid 92 is threonine, amino acid 125 is leucine, amino acid 147 is threonine, amino acid 162 is asparagine, and amino acid 196 is aspartate. In some embodiments, an improved RFP comprises the amino acid sequence of SEQ ID NO: 15 (iRFD1), SEQ ID NO: 17 (iRFD2), or SEQ ID NO: 19 (iRFD3). In some embodiments, an improved RFP consists of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19. Any of the described improved RFPs can be provided as a fusion molecule. In some embodiments, the fusion molecule comprises the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19. The im