Yeast promoters from pichia pastoris

Abstract

In accordance with the invention, isolated nucleic acids, expression methods, host cells, expression vectors, and DNA constructs for producing proteins, and proteins produced using the expression methods are described. More particularly, nucleic acids isolated from Pichia pastoris wherein the nucleic acids have promoter activity are described. The invention also relates to expression methods, host cells, expression vectors, and DNA constructs, for using the Pichia pastoris promoters to produce proteins, and to the proteins produced using the expression methods.

1 - 45 . (canceled) 
     
     
         46 . An isolated nucleic acid comprising the sequence of a constitutive  Pichia pastoris  promoter, wherein the isolated nucleic acid is operably linked to a heterologous coding sequence, wherein the sequence of the isolated nucleic acid comprises a sequence at least 95% identical to a fragment of SEQ ID NO:2 wherein the fragment comprises at least 222 nucleotides upstream from the 3′ end of SEQ ID NO:2, wherein the fragment is a continuous fragment of SEQ ID NO:2, and wherein the fragment comprises a TATA box sequence to direct initiation of transcription. 
     
     
         47 . The isolated nucleic acid of  claim 46  wherein the sequence of the isolated nucleic acid is at least 98% identical to the fragment of SEQ ID NO:2. 
     
     
         48 . The isolated nucleic acid of  claim 46  wherein the sequence of the isolated nucleic acid is the fragment of SEQ ID NO:2. 
     
     
         49 . The isolated nucleic acid of  claim 46  wherein the heterologous coding sequence encodes a protein selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein. 
     
     
         50 . The isolated nucleic acid of  claim 46  wherein the heterologous coding sequence encodes an enzyme for use in animal feed. 
     
     
         51 . The isolated nucleic acid of  claim 50  wherein the enzyme is selected from the group consisting of a mannanase, an amylase, a glucanase, a protease, a phytase, a galactosidase, a cellulase, and a xylanase. 
     
     
         52 . The isolated nucleic acid of  claim 51  wherein the protein is a phytase. 
     
     
         53 . The isolated nucleic acid of  claim 51  wherein the protein is a galactosidase. 
     
     
         54 . An expression vector comprising the isolated nucleic acid of  claim 46 . 
     
     
         55 . A host cell comprising the expression vector of  claim 54 . 
     
     
         56 . A host cell comprising the isolated nucleic acid of  claim 46 . 
     
     
         57 . The host cell of  claim 56  wherein the host cell is a  Pichia  species. 
     
     
         58 . The host cell of  claim 57  wherein the  Pichia  species is  Pichia pastoris.    
     
     
         59 . A DNA construct comprising the isolated nucleic acid of  claim 46 . 
     
     
         60 . A method of producing a protein, the method comprising the step of
 culturing in a culture medium a host cell comprising a first expression cassette comprising the isolated nucleic acid of  claim 46  wherein the heterologous coding sequence encodes a protein, and wherein the culturing is done under conditions permitting expression of the protein.   
     
     
         61 . The method of  claim 60  wherein the protein is selected from the group consisting of a toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, a structural protein, an immunogenic protein, and a cell signaling protein. 
     
     
         62 . The method of  claim 60  wherein the protein is an enzyme for use in animal feed. 
     
     
         63 . The method of  claim 62  wherein the enzyme is selected from the group consisting of a mannanase, an amylase, a glucanase, a protease, a phytase, a galactosidase, a cellulase, and a xylanase. 
     
     
         64 . The method of  claim 63  wherein the enzyme is a phytase. 
     
     
         65 . The method of  claim 63  wherein the enzyme is a galactosidase.