Materials and methods for protein production
Inventors
Hoyt, Martin Andrew • Guo, Xiao • Shankar, Smita
Assignees
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Publication Number
US-12116699-B2
Publication Date
2024-10-15
Expiration Date
Abstract
This document relates to materials and methods for the production of protein. In one aspect, this document provides a nucleic acid construct including a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element includes a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668-734 relative to SEQ ID NO: 28.
Core Innovation
The invention relates to nucleic acid constructs comprising a first alcohol oxidase promoter element in which the first alcohol oxidase promoter element comprises a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668-734 relative to SEQ ID NO: 28. The promoter element is configured as an alcohol oxidase 1 promoter element, and the construct further includes a nucleotide sequence operably linked to the first alcohol oxidase promoter element.
The nucleic acid constructs are used for expression in a cell to produce a product by expressing the nucleotide sequence operably linked to the first alcohol oxidase promoter element. The encoded first protein is selected from antibody, enzyme, regulatory protein, peptide hormone, blood clotting protein, cytokine, cytokine inhibitor, and heme-binding protein, including leghemoglobin (LegH).
The constructs and methods are described for use in yeast cells, including methylotrophic yeast such as Pichia pastoris, Pichia methanolica, Candida boidinii, Hansenula polymorpha, and Torulopsis. The content further states that product titer can be increased compared with promoter variants lacking the mutations, and that high expression can occur from pAOX1 in the absence of added methanol.
Claims Coverage
Two independent claims are directed to a nucleic acid construct with a mutated alcohol oxidase promoter element and to a method of producing a product in a cell by expressing that construct. Across the claim set, the core inventive theme is the mutation of the AOX1 promoter region spanning nucleotide positions 668-734 relative to SEQ ID NO: 28, with construct-based expression control and product formation in a cell.
Alcohol oxidase promoter mutation positions 668-734 relative to SEQ ID NO: 28
A nucleic acid construct comprising a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element comprises a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668-734 relative to SEQ ID NO: 28.
Expressing an operably linked mutated alcohol oxidase promoter construct in a cell to produce a product
A method of producing a product in a cell comprising expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element comprises a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668-734 relative to SEQ ID NO: 28.
The independent claims cover nucleic acid constructs and cell-based production in which a first alcohol oxidase promoter element carries one or more mutations at nucleotide positions 668-734 relative to SEQ ID NO: 28, controlling expression of an operably linked nucleotide sequence for product formation.
Stated Advantages
High expression can occur from pAOX1 in the absence of added methanol.
Increasing product titer compared with promoter variants lacking the mutations.
Increased expression of coding sequences operably linked to the mutated AOX1 promoter.
Expression can be performed potentially without added methanol.
Documented Applications
Recombinant protein production in methylotrophic yeast using engineered AOX1/pAOX1 promoter elements.
Producing a product in a cell by expressing a nucleic acid construct with a nucleotide sequence operably linked to a mutated alcohol oxidase promoter element.
Expression in yeast cells, including methylotrophic yeast such as Pichia pastoris, Pichia methanolica, Candida boidinii, Hansenula polymorpha, and Torulopsis.
Producing heterologous/exogenous proteins, including heme-binding proteins such as leghemoglobin variants and leghemoglobin (LegH), antibody, enzyme, regulatory protein, peptide hormone, blood clotting protein, cytokine, and cytokine inhibitor.
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