US 12,264,319 B2
Yeast promotors for protein expression
Ilya I. Tolstorukov, Claremont, CA (US); James M. Cregg, Claremone, CA (US); Thomas G. Chappell, San Marcos, CA (US); and Knut R. Madden, Carlsbad, CA (US)
Assigned to BIOGRAMMATICS, INC., Carlsbad, CA (US)
Filed by BIOGRAMMATICS, INC., Carlsbad, CA (US)
Filed on Jan. 4, 2023, as Appl. No. 18/149,872.
Application 18/149,872 is a continuation of application No. 16/861,384, filed on Apr. 29, 2020, granted, now 11,555,194.
Application 16/861,384 is a continuation of application No. 14/773,482, granted, now 10,676,750, issued on Jun. 9, 2020, previously published as PCT/US2014/022086, filed on Mar. 7, 2014.
Claims priority of provisional application 61/775,029, filed on Mar. 8, 2013.
Prior Publication US 2023/0340507 A1, Oct. 26, 2023
Int. Cl. C12N 15/81 (2006.01); C07K 14/39 (2006.01); C12N 9/40 (2006.01); C12P 21/02 (2006.01)
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.