Novel sulfurylase-luciferase fusion proteins and thermostable sulfurylase

Abstract

The present invention relates to the field of DNA recombinant technology. More specifically, this invention relates to fusion proteins comprising an ATP generating polypeptide joined to a polypeptide that converts ATP into a detectable entity. Accordingly, this invention focuses on sulfurylase-luciferase fusion proteins. This invention also relates to pharmaceutical compositions containing the fusion proteins and methods for using them.

1 - 221 . (canceled)  
     
     
         222 . A method of determining the base sequence of a plurality of single stranded template nucleotides on an array, the method comprising: 
 (a) providing a planar surface comprises at least 400,000 discrete cavities, wherein each cavity forms a reaction chamber containing single-stranded nucleic acid templates of a single species, wherein the reaction chambers have a center to center spacing of between 5 to 200 μm, wherein each reaction chamber contains a reaction mixture comprising a template-directed nucleotide polymerase and said one of said plurality of single-stranded template nucleotides hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the single-stranded template nucleotides to form at least one unpaired nucleotide residue in each template at the 3′-end of the primer strand;    (b) adding an activated nucleotide 5′-triphosphate precursor of one known nitrogenous base to the reaction chambers under conditions which allow incorporation of the activated nucleoside 5′-triphosphate precursor onto the 3′-end of the primer strand, provided the nitrogenous base of the activated nucleoside 5′-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;    (c) detecting whether or not the nucleoside 5′-triphosphate precursor was incorporated into the primer strands in each reaction chamber by detecting a sequencing byproduct with an ATP generating polypeptide-ATP converting polypeptide fusion protein or an ATP generating protein and an ATP converting protein, thus indicating that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5′-triphosphate precursor in each reaction chamber;    (d) sequentially repeating steps (b) and (c), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5′-triphosphate precursor of known nitrogenous base composition; and    (e) determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of said nucleoside precursors.    
     
     
         223 . The method of  claim 222  wherein said sequencing byproduct is pyrophosphate.  
     
     
         224 . The method of  claim 222  wherein the ATP generating polypeptide-ATP converting polypeptide fusion protein comprises an ATP generating polypeptide portion with an amino acid sequence which is at least 96% homologous to SEQ ID NO:2.  
     
     
         225 . The method of  claim 222  wherein the ATP generating polypeptide-ATP converting polypeptide fusion protein comprises an ATP generating polypeptide portion with an amino acid sequence which is SEQ ID NO:6.  
     
     
         226 . The method of  claim 222  wherein the ATP generating polypeptide-ATP converting polypeptide fusion protein comprises an amino acid sequence of SEQ ID NO:4.  
     
     
         227 . The method of  claim 222  wherein the ATP generating protein comprises an amino acid sequence which is at least 96% homologous to SEQ ID NO:2.  
     
     
         228 . The method of  claim 222  wherein the ATP generating protein comprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6.  
     
     
         229 . The method of  claim 222  wherein said ATP generating polypeptide-ATP converting polypeptide fusion protein comprise an amino acid sequence encoded by a polynucleotide with an open reading frame of SEQ ID NO:3.  
     
     
         230 . The method of  claim 222  wherein said ATP generating polypeptide comprise an amino acid sequence encoded by a polynucleotide with an open reading frame which is no more than 11% different from an open reading frame of SEQ ID NO:1.  
     
     
         231 . The method of  claim 222  wherein said ATP generating polypeptide comprises an amino acid sequence encoded by an open reading frame of SEQ ID NO:1 or SEQ ID NO:5.  
     
     
         232 . The method of  claim 222  wherein said ATP generating polypeptide-ATP converting polypeptide fusion protein or said ATP generating protein further comprises an affinity tag.  
     
     
         233 . The method of  claim 222  wherein said ATP generating polypeptide-ATP converting polypeptide fusion protein, said ATP generating protein, or said ATP converting polypeptide is bound to a bead.  
     
     
         234 . A method of identifying a base at a target position in a sample nucleic acid sequence, comprising providing a sample nucleic acid and a primer which hybridizes to the sample nucleic acid immediately adjacent to the target position, subjecting the sample nucleic acid and primer to a polymerase reaction in the presence of a nucleotide whereby the nucleotide will only become incorporated if it is complementary to the base in the target position, and detecting said incorporation of the nucleotide by monitoring the release of inorganic pyrophosphate, whereby detection of incorporation of said nucleotide is indicative of identification of a base at a target position that is complementary to said nucleotide, and wherein the release of inorganic pyrophosphate is detected using a thermostable sulfurylase-luciferase fusion protein or a thermostable sulfurylase.  
     
     
         235 . The method of  claim 234  wherein the thermostable sulfurylase-luciferase fusion protein or the thermostable sulfurylase comprises an amino acid of at least 96% homology to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.  
     
     
         236 . The method of  claim 234  wherein the thermostable sulfurylase-luciferase fusion protein or the thermostable sulfurylase is encoded by an open reading frame of SEQ ID NO: 1, 3 or 5.  
     
     
         237 . The method of  claim 234  wherein the thermostable sulfurylase-luciferase fusion protein or the thermostable sulfurylase further comprises an affinity tag.  
     
     
         238 . The method of  claim 234  wherein said the thermostable sulfurylase-luciferase fusion protein or the thermostable sulfurylase is bound to a bead.