CPC C12N 15/815 (2013.01) [C07K 14/39 (2013.01); C12N 9/2465 (2013.01); C12P 21/02 (2013.01); C12N 2800/60 (2013.01); C12Y 302/01022 (2013.01)] | 8 Claims |
1. A method of producing a protein, wherein the protein is expressed using a first expression cassette in combination with a second expression cassette, wherein the method comprises the step of culturing in a culture medium a host cell comprising a first expression cassette comprising
a promoter sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 14, or at least 95% identical to a fragment thereof, wherein the promoter sequence comprises the sequence of a constitutive Pichia pastoris promoter; and
a heterologous coding sequence, wherein said promoter sequence is operably linked to said heterologous coding sequence, and
a second expression cassette comprising
(a) said heterologous coding sequence encoding the protein operably linked to a second nucleic acid, said second nucleic acid comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 4, wherein SEQ ID NO: 2 or SEQ ID NO: 4 have promoter activity; or
(b) said heterologous coding sequence encoding the protein operably linked to a second nucleic acid, said second nucleic acid comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 15 or SEQ ID NO: 16, wherein SEQ ID NO: 15 or SEQ ID NO: 16 have promoter activity; or
(c) said heterologous coding sequence encoding the protein operably linked to a different AOX promoter than SEQ ID NO: 15 or SEQ ID NO: 16, an FLD promoter, or a DAS promoter, wherein said promoter and second nucleic acid each comprise a TATA box sequence to direct initiation of transcription, said heterologous coding sequence encodes a protein, and wherein the culturing is done under conditions permitting expression of the protein.
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