Long wavelength engineered fluorescent proteins

Abstract

Engineered fluorescent proteins, nucleic acids encoding them and methods of use.

1 .- 32 . (canceled) 
     
     
         33 . A fluorescently labeled antibody comprising an antibody coupled to a functional engineered fluorescent protein whose amino acid sequence is substantially identical to the amino acid sequence of  Aequorea  green fluorescent protein (SEQ ID NO: 2) and which differs from SEQ ID NO: 2 by at least the amino acid substitution T203X, wherein X is an aromatic amino acid selected from H, Y, W or F, said functional engineered fluorescent protein having a different fluorescent property than  Aequorea  green fluorescent protein. 
     
     
         34 . The fluorescently labeled antibody of  claim 33  wherein the amino acid sequence further comprises a substitution at S65, wherein the substitution is selected from S65G, S65T, S65A, S65L, S65C, S65V and S65I. 
     
     
         35 . The fluorescently labeled antibody of  claim 33  wherein the amino acid sequence differs by no more than the substitutions S65T/T203H; S65T/T203Y; S72A/F64L/S65G/T203Y; S72A/S65G/V68L/T203Y; S65G/V68L/Q69K/S72A/T203Y; S65G/S72A/T203Y; or S65G/S72A/T203W. 
     
     
         36 . The fluorescently labeled antibody of  claim 33  or  34  wherein the amino acid sequence further comprises a substitution at Y66, wherein the substitution is selected from Y6611, Y66F, and Y66W. 
     
     
         37 . The fluorescently labeled antibody of any of  claims 33 - 35  which is a fusion protein wherein the fusion protein comprises the antibody fused to the functional engineered fluorescent protein. 
     
     
         38 . A nucleic acid molecule comprising a nucleotide sequence encoding an antibody fused to a nucleotide sequence encoding a functional engineered fluorescent protein whose amino acid sequence is substantially identical to the amino acid sequence of  Aequorea  green fluorescent protein (SEQ ID NO: 2) and which differs from SEQ ID NO: 2 by at least the amino acid substitution T203X, wherein X is an aromatic amino acid selected from H, Y, W or F, said functional engineered fluorescent protein having a different fluorescent property than  Aequorea  green fluorescent protein. 
     
     
         39 . The nucleic acid molecule of  claim 38  wherein the amino acid sequence further comprises a substitution at S65, wherein the substitution is selected from S65G, S65T, S65A, S65L, S65C, S65V and S65I. 
     
     
         40 . The nucleic acid molecule of  claim 38  wherein the amino acid sequence differs by no more than the substitutions S65T/T203H; S65T/T203Y; S72A/F64L/S65G/T203Y; S72A/S65G/V68L/T203Y; S65G/V68L/Q69K/S72A/T203Y; S65G/S72A/T203Y; or S65G/S72A/T203W. 
     
     
         41 . The nucleic acid molecule of  claim 38  or  39  wherein the amino acid sequence further comprises a substitution at Y66, wherein the substitution is selected from Y66H, Y66F, and Y66W. 
     
     
         42 - 53 . (canceled) 
     
     
         54 . A fluorescently labeled antibody comprising an antibody coupled to a functional engineered fluorescent protein whose amino acid sequence is substantially identical to the amino acid sequence of  Aequorea  green fluorescent protein (SEQ ID NO: 2) and which differs from SEQ ID NO: 2 by at least an amino acid substitution at L42, V61, T62, V68, Q69, Q94, N121, Y145, H148, V150, F165, I167, Q183, N185, L220, E222 (not E222G), or V224, said functional engineered fluorescent protein having a different fluorescent property than  Aequorea  green fluorescent protein. 
     
     
         55 . The antibody of  claim 54  wherein the amino acid substitution is:
 L42X, wherein X is selected from C, F, H, W and Y,   V61X, wherein X is selected from F, Y, H and C,   T62X, wherein X is selected from A, V, F, S, D, N, Q, Y, H and C,   V68X, wherein X is selected from F, Y and H,   Q69X, wherein X is selected from K, R, E and G,   Q94X, wherein X is selected from D, E, H, K and N,   N121X, wherein X is selected from F, H, W and Y,   Y145X, wherein X is selected from W, C, F, L, E, H, K and Q,   H148X, wherein X is selected from F, Y, N, K, Q and R,   V150X, wherein X is selected from F, Y and H,   F165X, wherein X is selected from H, Q, W and Y,   I167X, wherein X is selected from F, Y and H,   Q183X, wherein X is selected from H, Y, E and K,   N185X, wherein X is selected from D, E, H, K and Q,   L220X, wherein X is selected from H, N, Q and T,   E222X, wherein X is selected from N and Q or   V224X, wherein X is selected from H, N, Q, T, F, W and Y.   
     
     
         56 . A nucleic acid molecule comprising a nucleotide sequence encoding an antibody fused to a nucleotide sequence encoding a functional engineered fluorescent protein whose amino acid sequence is substantially identical to the amino acid sequence of  Aequorea  green fluorescent protein (SEQ ID NO: 2) and which differs from SEQ ID NO: 2 by at least an amino acid substitution at L42, V61, T62, V68, Q69, Q94, N121, Y145, H148, V150, F165, I167, Q 183, N185, L220, E222 (not E222G), or V224, said functional engineered fluorescent protein having a different fluorescent property than  Aequorea  green fluorescent protein.