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Kluyveromyces marxianus derived high-expression promoter and expression system thereof

A high-expression and promoter technology, applied in the field of microbial genetic engineering applications, can solve the problems of limited expression vectors, achieve high transcriptional activity, strong transcriptional activation activity, and fast and convenient screening effects

Active Publication Date: 2016-04-27
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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  • Kluyveromyces marxianus derived high-expression promoter and expression system thereof
  • Kluyveromyces marxianus derived high-expression promoter and expression system thereof
  • Kluyveromyces marxianus derived high-expression promoter and expression system thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] (1) Acquisition of KmINU1 promoter

[0046] The genome of Kluyveromyces scicerisporus (Kluyveromycescicerisporus) CBS4857 is used as a template, and Inu-1058 (SEQ ID No.11) and Inu-0 (SEQ ID No.13) are used as primers, and are amplified by polymerase chain reaction (PCR) The KmlINU1 promoter was obtained, its nucleotide sequence is shown in SEQ ID No.1, and its total length is 1058bp.

[0047] The genome of Kluyveromyces scicerisporus (Kluyveromycescicerisporus) 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 to amplify by polymerase chain reaction (PCR). KmlINU1 promoter was obtained by increasing method, its nucleotide sequence is shown in SEQ ID No.2, and its total length is 367bp.

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

[0049] The genome of Kluyveromyces scicerisporus (Kluyveromycescicerisporus) CBS4857 was used as a template, inu-PS-F (SEQ ID No.5) and inu-PS-R (SEQ ID No...

Embodiment 2

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

[0053] The KmINU1 promoter of SEQIDNo.1 that embodiment 1 obtains is truncated into 5 sections, as Figure 6 shown. Figure 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 SEQIDNo.2) and KmPGK promoter activity comparison

[0063] In order to compare the most ideal KmINU1 promoter (shown in SEQIDNo.2) proposed by the present invention and the relationship between the activity of the strongest KmPGK promoter of Kluyvernia reported at present, Kluyveromyces lactis is used as the expression host , GFP as the reporter gene for experiments. The specific experimental steps are as follows:

[0064] (1) Using the plasmid pCXJ10 as the basic backbone (GenBankU34314), 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 by T4 ligase, and the ligated product is t...

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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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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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