Recombinant Colwellia Psychrerythraea Alkaline Phosphatase and Uses Thereof

Abstract

A heat labile alkaline phosphatase enzyme and methods of using the same and kits including the same are disclosed. Specifically, a nucleotide sequence of, peptide sequence of, methods of using, and kits comprising, a heat labile alkaline phosphatase isolated from Colwellia psychrerythraea are provided. Methods of over-expression and purification of the recombinant alkaline phosphatase and mutants thereof are also disclosed. Methods of over-expressing and purifying commercially useful quantities of active recombinant heat labile alkaline phosphatase fusion enzymes from C. psychrerythraea, wherein the fusion enzymes comprise one or more heterologous leader sequences are disclosed. The disclosed C. psychrerythraea heat labile alkaline phosphatase has properties similar to shrimp alkaline phosphatase and can be substituted for shrimp alkaline phosphatase in assays involving the same.

1 . An isolated nucleic acid comprising:
 a) a recombinant sequence which is at least 95% identical to SEQ ID NO:3;   b) the sequence which is fully complementary to SEQ ID NO:3;   c) the sequence which is fully complementary to the sequence which is at least 95% identical to SEQ ID NO:3; or   d) the sequence which hybridizes to SEQ ID NO:3 under stringent conditions.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . An isolated recombinant protein comprising the sequence according to SEQ ID NO:15, or the sequence at least 95% identical to SEQ ID NO:15. 
     
     
         5 - 6 . (canceled) 
     
     
         7 . A method of dephosphorylating a compound comprising a phosphate group, which comprises incubating a compound comprising a phosphate group with an effective amount of an isolated enzyme encoded by:
 a) the sequence according to SEQ ID NO:3;   b) a sequence which is at least 95% identical to SEQ ID NO:3;   c) the sequence which is fully complementary to SEQ ID NO:3;   d) the sequence which is fully complementary to the sequence which is at least 95% identical to SEQ ID NO:3; or   e) the sequence which hybridizes to SEQ ID NO:3 under stringent conditions.   
     
     
         8 . The method according to  claim 7 , wherein said compound is a nucleic acid, DNA or RNA. 
     
     
         9 . The method according to  claim 8 , further comprising incubating the nucleotide with an exonuclease prior to direct DNA sequencing. 
     
     
         10 . The method according to  claim 9 , performed immediately following termination of a polymerase chain reaction, wherein said nucleotide is an unused substrate of the polymerase chain reaction. 
     
     
         11 . The method according to  claim 7 , further comprising labeling dephosphorylated products from the incubation step with  32 P by further incubation with [γ- 32 P]NTP and an effective amount of T4 polynucleotide kinase. 
     
     
         12 - 15 . (canceled) 
     
     
         16 . A protein comprising a first peptide sequence coded from a nucleotide sequence selected from the group consisting of SEQ IDs NOs: 1-3 and nucleotide sequences at least 95% identical to one of SEQ IDs NOs: 1-3, and a second peptide sequence consisting of from one to ten repeats of the dipeptide HQ. 
     
     
         17 . The protein of  claim 16 , said second peptide sequence being fused to the C-terminus of said first peptide sequence. 
     
     
         18 . A nucleic acid coding the protein of  claim 16 . 
     
     
         19 . A protein comprising from one to ten repeats of the dipeptide sequence HQ fused to either the N-terminus or the C-terminus of SEQ ID NO:15 or to a peptide sequence at least 90% identical to SEQ ID NO:15. 
     
     
         20 . The protein of  claim 19 , said one to ten repeats of said dipeptide sequence being fused to said C-terminus. 
     
     
         21 . A fusion nucleic acid comprising SEQ ID NO:3 having fused to the 3′-terminus thereof a nucleotide sequence coding a hydrophilic stretch of amino acids, said fusion nucleic acid being effective to yield large quantities of active heat-labile  C. psychrerythraea  alkaline phosphatase enzyme via expression from  E. coli  of a vector having said fusion nucleic acid inserted therein. 
     
     
         22 . A method of expressing a protein of interest, comprising fusing to a nucleic acid sequence coding said protein of interest a further nucleic acid sequence coding from one to ten repeats of the dipeptide HQ to yield a fusion nucleic acid, and expressing said fusion nucleic acid to yield said protein of interest in a large quantity and in active form. 
     
     
         23 . The method of  claim 22 , wherein said fusion nucleic acid is inserted into an expression vector and expressed therefrom.