LUCIGEN YELLOW (LucY), A YELLOW FLUORESCENT PROTEIN

Abstract

Described herein are isolated polynucleotides that encode a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74. Also described are expression constructs containing the polynucleotides, transformed host cells containing the expression constructions, the encoded fluorescent proteins themselves, and methods of using the nucleotides and encoded fluorescent proteins for bioanalytical research.

What is claimed is: 
     
         1 . A recombinant polynucleotide comprising a nucleotide sequence encoding a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74. 
     
     
         2 . The recombinant polynucleotide of  claim 1 , wherein the nucleotide sequence further encodes at least one additional polypeptide of interest in-frame with the encoded protein. 
     
     
         3 . The recombinant polynucleotide of  claim 2 , wherein the at least one additional polypeptide of interest comprises a tag. 
     
     
         4 . The recombinant polynucleotide of  claim 3 , wherein the tag comprises a poly-His tag. 
     
     
         5 . The recombinant polynucleotide of  claim 1 , wherein the encoded protein is thermostable. 
     
     
         6 . The recombinant polynucleotide of  claim 1 , wherein fluorescence of the encoded protein at about 50° C. is greater than about 10% of fluorescence of the encoded protein at about 30° C. 
     
     
         7 . The recombinant polynucleotide of  claim 1 , wherein fluorescence of the encoded protein at about 60° C. is greater than about 10% of fluorescence of the encoded protein at about 30° C. 
     
     
         8 . The recombinant polynucleotide of  claim 1 , wherein fluorescence of the encoded protein at about 70° C. is greater than about 10% of fluorescence of the encoded protein at about 30° C. 
     
     
         9 . The recombinant polynucleotide of  claim 1 , wherein fluorescence of the encoded protein at about 75° C. is greater than about 10% of fluorescence of the encoded protein at about 30° C. 
     
     
         10 . The recombinant polynucleotide of  claim 1 , wherein the nucleotide sequence is optimized to express the fluorescent protein in a heterologous host. 
     
     
         11 . The recombinant polynucleotide of  claim 1 , wherein the nucleotide sequence encodes a fluorescent protein which is at least 95% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74. 
     
     
         12 . An expression construct comprising a recombinant polynucleotide as recited in  claim 1 . 
     
     
         13 . A host cell comprising an expression construct as recited in  claim 12 . 
     
     
         14 . A method for making a fluorescent protein comprising cultivating the host cell of  claim 13 . 
     
     
         15 . A fluorescent protein made by cultivating a host cell, wherein the host cell comprises an expression construct, the expression construct comprises a recombinant polynucleotide, and the recombinant polynucleotide comprises a nucleotide sequence encoding a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74. 
     
     
         16 . A fusion protein comprising a polypeptide of interest operationally linked to a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74. 
     
     
         17 . A method of increasing the aqueous solubility of a protein of interest comprising fusing or attaching the protein of interest to a protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74. 
     
     
         18 . A method of assaying gene expression comprising:
 cultivating a host cell comprising an expression construct, wherein the expression construct comprises a recombinant polynucleotide and the recombinant polynucleotide comprises a nucleotide sequence encoding a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74; and   detecting fluorescence in the cell or an extract thereof.