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Application of Hot1p as positive regulatory factor in improvement of protein expression in host cells

A host cell and protein expression technology, which is applied in the fields of molecular biology and bioengineering to achieve the effect of improving expression intensity, high-efficiency expression, and improving expression efficiency

Active Publication Date: 2022-07-22
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on its transcriptional regulation of Pgap

Method used

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  • Application of Hot1p as positive regulatory factor in improvement of protein expression in host cells
  • Application of Hot1p as positive regulatory factor in improvement of protein expression in host cells
  • Application of Hot1p as positive regulatory factor in improvement of protein expression in host cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Construction of transcription regulator Hot1p expression cassette

[0022] 1. Amplification of the Pgap promoter

[0023] The present invention takes the Pgap sequence of Pichia pastoris as a reference, uses software DNAMAN 8 to design and synthesize two oligonucleotide primers, and amplifies the Pgap target gene by PCR method.

[0024] The two PCR primers are as follows:

[0025] 1-F:ACTTAC GAGCTC GAGATCTTTTTTGTAG (SEQ ID NO: 4)

[0026] 1-R:ACTTACGC GGATCC GCGATAGTTGTTCAATTG (SEQ ID NO: 5)

[0027] The underlined bases are SacI and BamHI restriction endonuclease sites, respectively.

[0028] The PCR reaction system is shown in Table 1 below.

[0029] Table 1:

[0030] component Volume (μL) 2×Q5 Master Mix 12.5 Primer F (10μM) 1.25 Primer R (10μM) 1.25 template DNA 1(100ng) ddH 2 O

9ul total capacity 25ul

[0031] The PCR program settings are shown in Table 2.

[0032] Table 2:

[0033] ...

Embodiment 2

[0063] Example 2: Integration of the Hot1 gene into the Pichia genome

[0064] In order to improve the integration efficiency of the monocope expression cassette on Pichia pastoris chromosomes, the expression cassette was linearized with restriction endonuclease Sal I and purified and recovered with the kit. The recipient bacteria in this experiment was Pichia pastoris SMD1168 (recombinant bacteria with xylanase xynB gene inserted after Pgap, EX6). identification.

[0065] The results of PCR product sequencing showed that the screened strains were positive clones that co-expressed Hot1p.

Embodiment 3

[0066] Example 3: Hot1p enhances heterologous protein expression assay driven by the constitutive promoter Pgap

[0067] The positive clone 1 screened in Example 1 and the positive clone 2 screened in Example 2 were fermented, and the fermentation conditions were 28° C., 200 rpm, and cultured for 72 h. At the same time, Pichia pastoris (EX6) containing xynB gene was used as a control, and the supernatant was collected after 72 hours of culture for SDS-PAGE electrophoresis detection, and the expression level of xylanase xynB was analyzed.

[0068] Compared with the control group, the expression level of clone 1 picked after overexpressing the Hot1p transcription factor was increased by 99%, the expression level of clone 2 was increased by 101% compared with the control group, and the expression level of clone 3 was increased compared with the control group. Compared with the control group, the expression level of clone 4 was increased by 129%, and the expression level of clone ...

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Abstract

The invention relates to application of a transcriptional regulation factor expressed by eukaryotic genes, in particular to application of a transcriptional regulation factor Hot1p of a constitutive promoter Pgap. The invention discloses application of Hot1p as a positive regulatory factor in improvement of protein expression in host cells. The amino acid sequence of the Hot1p is coded by an Hot1 gene with the nucleotide sequence of SEQ ID NO: 1; according to the application, an Hot1 gene is inserted behind a promoter Pgap, so that the expression of protein in host cells is improved. The application disclosed by the invention can enhance transcription of a constitutive promoter Pgap promoter of the pichia pastoris, so that a subsequent exogenous gene is efficiently expressed in the pichia pastoris, and the situation that the difference of expression quantities of different genes is too large due to a dilution effect generated by using the same promoter during multi-copy or multi-gene expression can be avoided.

Description

technical field [0001] The invention belongs to the fields of molecular biology and bioengineering, and relates to the application of a transcriptional regulator of eukaryotic gene expression, in particular to the application of a transcriptional regulator Hot1p of a constitutive promoter Pgap. Background technique [0002] The promoter is one of the most important elements in regulating gene expression, P AOX1 Promoter (inducible) and Pgap promoter (constitutive) are the most representative promoters for exogenous protein expression in Pichia pastoris. [0003] Methanol-inducible Pichia expression system is currently a commonly used expression system for expressing most heterologous proteins. However, not all exogenous proteins are suitable for methanol-inducible expression and methanol has great safety hazards in large-scale production. Compared with methanol induction, the constitutive Pichia pastoris expression system does not use methanol induction when expressing hete...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/39C07K14/395C07K14/40C12N15/81C12N15/67C12N15/113C12N1/19C12R1/84C12R1/865C12R1/72
CPCC07K14/39C07K14/395C07K14/40C12N15/81C12N15/815C12N15/67C12N2800/102Y02A50/30
Inventor 姚冬生林香娜刘大岭谢春芳丁伟秋
Owner JINAN UNIVERSITY
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