Oplophorus-derived luciferases, novel coelenterazine substrates, and methods of use

A polynucleotide encoding an OgLuc variant polypeptide having at least 60% amino acid sequence identity to SEQ ID NO:

• 1 comprising at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID NO:
• 1 wherein the OgLuc variant polypeptide has enhanced luminescence relative to an OgLuc polypeptide of SEQ ID NO:
• 3 with the proviso that the polypeptide encoded by the polynucleotide is not one of those listed in Table 47.

The polynucleotide of 52, wherein the encoded OgLuc variant polypeptide further comprises amino acid substitutions V21L, F68Y, L72Q, M75K, H92R and V158I corresponding to SEQ ID NO:

• 1.

The polynucleotide of 55, wherein the amino acid at position 21 is leucine, at position 68 is tyrosine, at position 72 is glutamine, at position 75 is lysine, at position 92 is arginine, and at position 158 is isoleucine corresponding to SEQ ID NO:

• 1.

A polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to an OgLuc polypeptide of SEQ ID NO:

• 1 comprising amino acid substitutions A4E, Q11R, A33K, V44I, P115E, Q124K, Y138I, N166R, I90V, F541, Q18L, F68Y, L72Q, and M75K corresponding to SEQ ID NO:
• 1 and the OgLuc variant polypeptide having luciferase activity.

A polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to an OgLuc polypeptide of SEQ ID NO:

• 1, wherein the amino acid at position 4 is glutamate, at position 11 is arginine, at position 18 is leucine, at position 33 is lysine, at position 44 is isoleucine, at position 54 is isoleucine, at position 68 is tyrosine, at position 72 is glutamine, at position 75 is lysine, at position 90 is valine, at position 115 is glutamate, at position 124 is lysine, at position 138 is isoleucine, and at position 166 is arginine corresponding to SEQ ID NO:
• 1 and the OgLuc variant polypeptide having luciferase activity.

A polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to an OgLuc polypeptide of SEQ ID NO:

• 1 comprising amino acid substitutions A4E, Q11R, A33K, V44I, P115E, Q124K, Y138I, N166R, Q18L, F541, L92H, and Y109F corresponding to SEQ ID NO:
• 1 and the OgLuc variant polypeptide having luciferase activity.

A polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to an OgLuc polypeptide of SEQ ID NO:

• 1 comprising amino acid substitutions A4E, Q11R, A33K, V44I, A541, F77Y, I90V, P115E, Q124K, Y1381 and N166R corresponding to SEQ ID NO:
• 1 and the OgLuc variant polypeptide having luciferase activity.

A polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to an OgLuc polypeptide of SEQ ID NO:

• 1, wherein the amino acid at position 4 is glutamate, at position 11 is arginine, at position 18 is leucine, at position 33 is lysine, at position 44 is isoleucine, at position 54 is isoleucine, at position 92 is histidine, at position 109 is phenylalanine, at position 115 is glutamate, at position 124 is lysine, at position 138 is isoleucine, and at position 166 is arginine corresponding to SEQ ID NO:
• 1 and the OgLuc variant polypeptide having luciferase activity.

A polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to an OgLuc polypeptide of SEQ ID NO:

• 1, wherein the amino acid at position 4 is glutamate, at position 11 is arginine, at position 33 is lysine, at position 44 is isoleucine, at position 54 is isoleucine, at position 77 is tyrosine, at position 90 is valine, at position 115 is glutamate, at position 124 is lysine, at position 138 is isoleucine, and at position 166 is arginine corresponding to SEQ ID NO:
• 1 and the OgLuc variant polypeptide having luciferase activity.

A polynucleotide comprising the polynucleotide encoding the polypeptide of SEQ ID NO:

• 19.

An isolated polynucleotide encoding a decapod luciferase polypeptide having at least 30% amino acid sequence identity to SEQ ID NO:

• 1, the polypeptide comprising a sequence pattern corresponding to the sequence pattern of Formula (VII) and including no more than 5 differences, wherein differences include differences from pattern positions 1, 2, 3, 5, 8, 10, 12, 14, 15, 17, or 18 relative to Formula (VII) according to the OgLuc pattern listed in Table 4 as well as gaps or insertions between any of the pattern positions of Formula (VII) according to the OgLuc pattern listed in Table 4, wherein the decapod luciferase produces luminescence in the presence of a coelenterazine.

The vector of claim 96, wherein the polynucleotide is operably linked to a promoter.

A synthetic nucleotide sequence encoding an OgLuc variant polypeptide comprising a fragment of at least 300 nucleotides having 80% or less nucleic acid sequence identity to a parent nucleic acid sequence having SEQ ID NO:

• 14 and having 90% or more nucleic acid sequence identity to SEQ ID NO:
• 22 or SEQ ID NO:
• 23 or the complement thereof, wherein the decreased sequence identity is a result of different codons in the synthetic nucleotide sequence relative to the codons in the parent nucleic acid sequence, wherein the synthetic nucleotide sequence encodes a firefly luciferase which has at least 85% amino acid sequence identity to the corresponding luciferase encoded by the parent nucleic acid sequence, and wherein the synthetic nucleotide sequence has a reduced number of regulatory sequences relative to the parent nucleic acid sequence.

A synthetic nucleotide sequence encoding an OgLuc variant polypeptide comprising a fragment of at least 100 nucleotides having 80% or less nucleic acid sequence identity to a parent nucleic acid sequence having SEQ ID NO:

• 18 and having 90% or more nucleic acid sequence identity to SEQ ID NO:
• 24 or SEQ ID NO:
• 25 or the complement thereof, wherein the decreased sequence identity is a result of different codons in the synthetic nucleotide sequence relative to the codons in the parent nucleic acid sequence, wherein the synthetic nucleotide sequence encodes a OgLuc variant which has at least 85% amino acid sequence identity to the corresponding luciferase encoded by the parent nucleic acid sequence, and wherein the synthetic nucleotide sequence has a reduced number of regulatory sequences relative to the parent nucleic acid sequence.

A fusion peptide comprising a signal peptide from Oplophorus gracilirostris fused to a heterologous protein, wherein said signal peptide is SEQ ID NO:

• 54, wherein the fusion peptide is expressed in a cell and secreted from the cell.

A method of generating a polynucleotide encoding a OgLuc variant polypeptide comprising:

• (a) using a parental fusion protein construct comprising a parental OgLuc polypeptide and at least one heterologous polypeptide to generate a library of variant fusion proteins;
• and
• (b) screening the library for at least one of enhanced luminescence, enhanced enzyme stability or enhanced biocompatibility relative to the parental fusion protein construct.

A method of generating codon-optimized polynucleotides encoding a luciferase for use in an organism, comprising:

• for each amino acid in the luciferase, randomly selecting a codon from the two most commonly used codons used in the organism to encode for the amino acid to produce a first codon-optimized polynucleotide.