C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 15/14 (2006.01) C07K 14/765 (2006.01) C12N 1/19 (2006.01) C12N 15/65 (2006.01) C12N 15/81 (2006.01)
Patent
CA 2058820
A novel expression cassette, vectors and process for the secretion of HSA in Pichia pastoris cells. In accordance with the present invention there has been discovered an improved expression cassette for the production of HSA in Pichia pastoris comprising a) a Pichia pastoris 5' regulatory region having a 5' end and a 3' end selected from the group consisting of the Pichia pastoris AOX1 5' regulatory region and the Pichia pastoris DAS1 5' regulatory region wherein the 3' end of the 5' regulatory region is operably linked to; b) an HSA structural gene encoding a signal sequence and mature protein having a 5' end and a 3' end wherein the HSA structural gene has an ATG start codon within about 11 deoxyribonucleotides of the 5' end of said HSA structural gene; and operably linked to c) a 3' termination sequence. In accordance with the present invention there has also discovered Pichia pastoris cells transformed with an improved expression cassette for the production of HSA in Pichia pastoris comprising a) a Pichia pastoris 5' regulatory region having a 5' end and a 3' end selected from the group consisting of the Pichia pastoris AOX1 5' regulatory region and the Pichia pastoris DAS1 5' regulatory region wherein the 3' end of the 5' regulatory region is operably linked to; b) an HSA structural gene encoding a signal sequence and mature protein having a 5' end and a 3' end wherein the HSA structural gene has an ATG start codon within about 11 deoxyribonucleotides of the 5' end of said HSA structural gene; and operably linked to c) a 3' termination sequence. In a further embodiment of the present invention, there has also discovered a process for the secretion of HSA from transformed Pichia pastoris cells comprising a) transforming a Pichia pastoris cell with at least one vector having at least one expression cassette comprising i) a Pichia pastoris 5' regulatory region having a 5' end and a 3' end selected from the group consisting of the Pichia pastoris AOX1 5' regulatory region and the Pichia pastoris DAS1 5' regulatory region wherein the 3' end of the 5' regulatory region is operably to ii) an HSA structural gene encoding a signal sequence and mature protein having a 5' end and a 3' end wherein the HSA structural gene said HSA structural gene; and operably linked to iii) a 3' termination sequence; and then b) culturing the resulting transformed Pichia pastoris suitable conditions to obtain the secretion of HSA.
Barr Kathryn A.
Brierley Russell A.
Sreekrishna Kotikanyad
Thill Gregory P.
Tschopp Juerg F.
Osler Hoskin & Harcourt Llp
Phillips Petroleum Company
Research Corporation Technologies Inc.
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