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A high-expression promoter derived from Kluyveromyces marx and its expression system

A high-expression, promoter technology, applied in microorganism-based methods, using vectors to introduce foreign genetic material, peptides, etc., can solve the problem of limited expression vectors, achieve high transcription activity, fast and convenient screening, and strong transcription initiation activity Effect

Active Publication Date: 2019-01-15
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with Saccharomyces cerevisiae and Pichia pastoris, Kluyveromyces is relatively limited as a host cell for expression vectors

Method used

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  • A high-expression promoter derived from Kluyveromyces marx and its expression system
  • A high-expression promoter derived from Kluyveromyces marx and its expression system
  • A high-expression promoter derived from Kluyveromyces marx and its expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] (1) Acquisition of KmINU1 promoter

[0046] Using the genome of Kluyveromyces cicerisporus CBS4857 as a template, Inu-1058 (SEQ ID No.11) and Inu-0 (SEQ ID No.13) as primers, the polymerase chain reaction ( PCR) amplification method to obtain the KmINU1 promoter, its nucleotide sequence is shown in SEQ ID No.1, and its total length is 1058bp.

[0047] The genome of Kluyveromyces cicerisporus (Kluyveromyces cicerisporus) CBS4857 was used as a template, and inu-PS-F (SEQ ID No.5) and Inu-0 (SEQ ID No.13) were used as primers. The KmINU1 promoter was obtained by reaction (PCR) amplification, its nucleotide sequence is shown in SEQ ID No. 2, and its total length is 367 bp.

[0048] (2) Acquisition of efficient exocrine signal peptide (SEQ ID No.3)

[0049] Using the genome of Kluyveromyces cicerisporus CBS4857 as a template, inu-PS-F (SEQ ID No.5) and inu-PS-R (SEQ ID No.6) as primers, polymerase The inulinase promoter was obtained by chain reaction (PCR) amplification, ...

Embodiment 2

[0052] 1. Differential analysis of the activation activity of KmINU1 promoters with different lengths

[0053] The KmINU1 promoter of SEQ ID No.1 that embodiment 1 obtains is truncated into 5 sections, as Image 6 shown. Image 6 In the above, the transcription start site (ATG) is defined as +1 position, the interval from -1058 to -1 is the KmINU1 promoter, and the interval from +1 to +69 is the high-efficiency exocrine signal peptide of the present invention. The KmlINU1 promoter starts from ATG and is truncated at -217bp, -367bp, -567bp, -817bp, -1058bp, respectively, to obtain KmlINU1 promoters with nucleotide sequence lengths of 217, 367, 567, 817 and 1058bp respectively sequence.

[0054] In order to study the differences in the promoter activities of the above-mentioned KmINU1 promoters of different lengths, and further determine their core promoter regions, the above-mentioned promoters of different lengths were linked to kanamycin resistance gene and reporter gene gr...

Embodiment 3

[0062] 367bp length KmINU1 promoter (shown in SEQ ID No.2) and KmPGK promoter activity comparison

[0063] In order to compare the most ideal KmINU1 promotor (shown in SEQ ID No.2) proposed by the present invention and the strong and weak relationship of the initiating activity of the Kluyveromyces strongest KmPGK promotor reported at present, take Kluyveromyces lactis as Expression host, green fluorescent protein as the reporter gene for experiments. The specific experimental steps are as follows:

[0064] (1) Using the plasmid pCXJ10 as the basic backbone (GenBank U34314), and according to the characteristics of the restriction site, select BamHI and EcoRI restriction sites to insert the kanamycin resistance gene (kanMX4). The kanMX4 fragment is derived from the double digestion product of plasmid pFA6a-kanMX4, the restriction site is BamHI and EcoRI, the digestion product is recovered and purified to obtain the kanMX4 DNA fragment, which is ligated with the vector pCXJ10 b...

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Abstract

The invention provides a Kluyveromyces marxianus derived high-expression promoter KmINU1 promoter, and an efficiently in vitro excreted signal peptide and an expression system thereof. The KmINU1 promoter having a nucleotide sequence represented by SEQ ID No.2 has strongest promotion activity, and has far higher activity than known KmPGK promoters. The KmINU1 promoter can start expression of a downstream gene without induction, and a target gene can efficiently express after stably growing in host cells, and is especially suitable for expression of exogenous genes influencing the growth of the host cells. The expression system containing the KmINU1 promoter and the efficiently in vitro excreted signal peptide, constructed in the invention, is suitable for various microzymes, such as Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus, and lays a foundation for efficient expression of different exogenous genes.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering applications, and specifically relates to a recombinant vector comprising a high-expression promoter derived from Kluyveromyces marx and an exocrine signal peptide, comprising the promoter and signal peptide, and utilizing the vector in various yeasts A method to achieve high expression of the target gene. Background technique [0002] The use of yeast cells for the secretion and expression of foreign proteins has been widely concerned. Yeast cells are widely used by researchers because of their advantages such as being suitable for large-scale production, a relatively complete post-translational modification system, and no endotoxin contamination. However, the same yeast cells also have the disadvantages of low expression level and incomplete signal peptide processing, which leads to the inability to secrete large proteins. At present, widely used and relatively mature yeast express...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C07K14/39C12N15/81C12N1/19C12R1/645
CPCC07K14/39C12N15/81C12N2800/102
Inventor 袁文杰高教琪李益民白凤武
Owner DALIAN UNIV OF TECH
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