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KR-20260064970-A - Artificial hybrid promoter

KR20260064970AKR 20260064970 AKR20260064970 AKR 20260064970AKR-20260064970-A

Abstract

The present invention relates to an artificial hybrid promoter, and more specifically, provides an artificial hybrid promoter comprising an upstream activation sequence of a GAL10 gene and a core promoter sequence of a PGK gene, a recombinant expression vector comprising the same, a host cell, and a method for producing a target protein using said host cell.

Inventors

  • 양선영

Assignees

  • 한화솔루션 주식회사

Dates

Publication Date
20260508
Application Date
20241030

Claims (9)

  1. An artificial hybrid promoter comprising the following (a) and (b): (a) upstream activation sequence (UAS) of the GAL10 gene; and (b) Core promoter sequence of the PGK gene.
  2. In claim 1, the UAS of the GAL10 gene is an artificial hybrid promoter that is repeatedly located upstream of the core promoter.
  3. An artificial hybrid promoter according to claim 1, wherein the UAS of the GAL10 gene comprises the nucleotide sequence of SEQ ID NO. 1, and the core promoter sequence of the PGK gene comprises the nucleotide sequence of SEQ ID NO. 2.
  4. In claim 1, the artificial hybrid promoter comprises the nucleotide sequence of SEQ ID NO. 3, the nucleotide sequence of SEQ ID NO. 4, the nucleotide sequence of SEQ ID NO. 5, or the nucleotide sequence of SEQ ID NO. 6.
  5. A recombinant expression vector comprising an artificial hybrid promoter of any one of claims 1 to 4.
  6. A recombinant expression vector according to claim 5, further comprising a gene encoding a target protein.
  7. A host cell comprising the recombinant expression vector of claim 5.
  8. In paragraph 7, the host cell is a yeast cell.
  9. A method for producing a target protein, comprising the step of culturing the host cells of claim 7 to produce the target protein.

Description

Artificial hybrid promoter The present invention relates to an artificial hybrid promoter, and more specifically, provides an artificial hybrid promoter comprising an upstream activation sequence of a GAL10 gene and a core promoter sequence of a PGK gene, a recombinant expression vector comprising the same, a host cell, and a method for producing a target protein using said host cell. Yeast-based protein expression and secretion systems offer several advantages in the production of recombinant proteins. As unicellular eukaryotes, yeast can be easily mass-cultured at a low cost. Unlike prokaryotic expression systems such as E. coli, yeast is capable of post-translational modifications, such as glycosylation, making it particularly suitable for the production of proteins derived from eukaryotic cells. Furthermore, unlike expression systems in E. coli, yeast enables soluble expression in almost all cases without forming inclusion bodies. Additionally, its well-developed protein secretion pathways ensure that subsequent processes are not complex, giving it high industrial value. To improve protein productivity in yeast, the use of high-expression promoters is indispensable. To date, various high-expression promoters have been developed to achieve high protein expression in yeast. Examples include constant high-expression promoters such as the promoter for the TDH3 gene (TDH3 promoter), the promoter for the PGK1 gene (PGK1 promoter), and the promoter for the ADH1 gene (ADH1 promoter), as well as inductive high-expression promoters such as the promoter for the GAL1 gene (GAL1 promoter) and the promoter for the GAL10 gene (GAL10 promoter). However, these natural promoters have limitations in transcriptional capacity, so hybrid promoters of a wider range of intensities are being developed to more precisely control gene expression by combining regulatory components and core components of various promoters. A traditional method for constructing a hybrid promoter involves combining the upstream activation sequence (UAS) of one promoter with the core sequence of another promoter. Generally, the UAS of the GAL promoter is used, and in Non-Patent Literature 1 , a UAS GAL10 -Core CYC1 hybrid promoter was reported by combining the UAS of the GAL10 promoter with the core sequence of the CYC1 promoter to express a heterologous protein in Saccharomyces cerevisiae. Additionally, in Non-Patent Literature 2, various hybrid promoters with different induction intensities were constructed by combining the UAS of the GAL1 promoter with the core sequence of another promoter. Against this background, the inventors completed the present invention by developing a novel artificial hybrid promoter capable of enhancing the expression of a target protein by at least 120% to a maximum of 330% compared to the existing GAL10 promoter by combining the UAS of the GAL10 promoter with the core sequence of the PGK promoter. Figure 1 shows a gene construct in which a UAS (GAL10), a PGK core promoter, and a gene encoding a target protein are sequentially linked. Figure 2 is a cleavage map of the UAS(GAL10)-PGK-MFα-TGFβ-GSlinker-RFP vector. Figure 3 is a cleavage map of the GAL10-MFα-TGFβ-GSlinker-RFP vector. Figure 4 compares the expression levels of the target protein in a strain transformed with the UAS(GAL10)-PGK-MFα-TGFβ-GSlinker-RFP vector and a strain transformed with the GAL10-MFα-TGFβ-GSlinker-RFP vector. Figure 5 is a cleavage map of the UAS(GAL10)-TDH3-MFα-TGF-RFP vector. Figure 6 is a cleavage map of the UAS(GAL10)-TEF1-MFα-TGF-RFP vector. Figure 7 compares the expression levels of target proteins with a hybrid promoter in which Pgal10 is combined with a tef1 core promoter or a tdh3 core promoter as another homeostatic promoter, and a UAS(GAL10)-PGK hybrid promoter in which Pgal10 UAS is linked with various repetitions (UAS(GAL10)-PGK, UAS(GAL10)×2-PGK, UAS(GAL10)×3-PGK, and UAS(GAL10)×4-PGK, respectively). Figure 8 is a cleavage map of the GAL10-MFα-RFP vector. Figure 9 is a cleavage map of the UAS(GAL10)-PGK-MFα-RFP vector. Figure 10 is a cleavage map of the UAS(GAL10)×3-PGK-MFα-RFP vector. The present invention will be described in more detail below. All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. All numbers expressing the size, quantity, and physical properties of a feature used in this specification and claims should be understood as being modified by the term "approximately" in all cases. Accordingly, unless otherwise indicated, the numerical parameters disclosed in this specification and claims are approximations that may vary depending on the desired properties to be obtained by a person skilled in the art using the teachings disclosed in this specificat