Synthetic luciferase gene and protein

Abstract

A codon optimized and stabilized luciferase gene and a novel recombinant DNA characterized by incorporating this new gene coding for a novel luciferase into a vector DNA for improved activities in mammalian cells, are disclosed. This new luciferase exhibits long-wavelength light emission, as well as improved thermostability and higher expression levels in mammalian cell systems, compared to native luciferase. Assays using this new enzyme for measuring various biological metabolic functions are described.

What is claimed is: 
     
         1 . An isolated nucleic acid molecule comprising a modified version of SEQ ID NO:1, wherein:
 A. such modifications consist of:
 i. alteration of at least one palindromic sequence, 
 ii. alteration of at least one cryptic splice acceptor site, 
 iii. alteration of at least one cis-acting motif, 
 iv. increasing GC content compared to native SEQ ID NO:1 by at least 10%, 
 v. removing at least one RNAse cleavage motif, and 
 vi. alteration of nucleotides 875-877 to encode an amino acid selected from the group consisting of Tyr, Lys, Leu and Gln. 
   B. wherein said isolated nucleic acid molecule encodes a protein that exhibits bioluminescence in the presence of D-luciferin at a wavelength between about 590 nm and about 620 nm.   
     
     
         2 . An isolated nucleic acid molecule encoding a COS luciferase protein comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:3 and a nucleic acid sequence having at least 90% identity to SEQ ID NO:3, wherein a protein encoded by said nucleic acid sequence exhibits bioluminescence in the presence of D-luciferin at a wavelength between about 590 nm and about 620 nm. 
     
     
         3 . An isolated nucleic acid molecule of  claim 2 , wherein said molecule encodes a protein comprising SEQ ID NO:4. 
     
     
         4 . A plasmid comprising an isolated nucleic acid molecule of  claim 2 . 
     
     
         5 . The plasmid of  claim 4 , wherein said plasmid contains one or more regulatory elements allowing expression in mammalian, bacterial or plant cells. 
     
     
         6 . The plasmid of  claim 4 , wherein said plasmid is selected from the group consisting of pCMV and pSV40. 
     
     
         7 . An isolated protein encoded by an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:3 and a nucleic acid sequence having at least 90% identity to SEQ ID NO:3, wherein a protein encoded by said nucleic acid sequence exhibits bioluminescence in the presence of D-luciferin at a wavelength between about 590 nm and about 620 nm. 
     
     
         8 . A method of producing the luciferase protein of  claim 7  comprising: culturing, in a medium, a microorganism belonging to the genus  Escherichia  having inserted therein a nucleic acid sequence encoding said protein and collecting the luciferase protein from the culture. 
     
     
         9 . A method for producing the luciferase protein according to  claim 8 , wherein said nucleic acid molecule is inserted into a plasmid DNA vector containing a Histidine tag and expressed in bacterial cells. 
     
     
         10 . A method for detecting ATP in a sample comprising:
 a. contacting an isolated protein of  claim 7  with a sample solution containing D-luciferin, one or more ATPase inhibitors and an unknown amount of ATP, and   b. measuring light emitted from said sample.   
     
     
         11 . The method of  claim 10 , further comprising the step of adding a known concentration of ATP to the sample. 
     
     
         12 . The method of  claim 10 , wherein the sample solution further comprises a cell lysing agent selected from the group consisting of Triton X-100, glycerol, TCA, DMSA, CTAB, and ethanol. 
     
     
         13 . The method of  claim 10 , wherein the sample solution further comprises NaF. 
     
     
         14 . The method of  claim 10 , wherein the sample solution further comprises an enzyme stabilizing agent selected from the group consisting of bovine serum albumin, gelatin, glycerol, ethylene glycol, polyethylene glycol and a detergent. 
     
     
         15 . A method of measuring cell viability within a sample population of cells by detecting ATP using the method of  claim 10 , wherein said sample solution further comprises a cell lysing reagent and a detergent and wherein the amount of light detected is proportional to the viability of the cells within the population. 
     
     
         16 . A method of measuring cell proliferation within a sample population of cells by detecting ATP using the method of  claim 10 , wherein said sample solution further comprises a cell lysing reagent and a detergent and wherein the amount of light detected is proportional to the cell growth and proliferation of the cells within the population. 
     
     
         17 . A method of measuring a second enzyme within a sample by detecting ATP using the method of  claim 10 , wherein said D-luciferin is a luciferin analog further comprises a pendant group specific for said second enzyme wherein the amount of light detected is proportional to the second enzyme concentration of the sample. 
     
     
         18 . The method of  claim 17 , wherein said solution further comprises a component selected from the group consisting of a cell lysing reagent, enzyme stabilizing reagent and a detergent. 
     
     
         19 . The method of  claim 17 , wherein said luciferin analog is selected from the group consisting of D-luciferin-6-O-β-D-glycopyranoside, D-luciferin-6-O amino acid, D-luciferin-4-O-amino acid, D-luciferin-6-O-methyl ether, D-luciferin-6-O-phosphate, D-luciferin-6-O-sulfate and D-luciferin-6-O-α-D-glycopyranoside. 
     
     
         20 . The method of  claim 17 , wherein said second enzyme is selected from the group consisting of β-glycosidase, a peptidase, a protease, cytochrome P450, alkaline phosphatase, acid phosphatase, aryl sulfatase, α-glycosidase and kinase.