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Method for improving expression level of recombinant protein in kluyveromyces

A Kluyveromyces and protein technology, applied in the direction of microorganism-based methods, recombinant DNA technology, botanical equipment and methods, etc., can solve the problems such as the unclear function of the Kluyveromyces lactis HAP1 gene, and achieve reduced production costs, The effect of increasing the amount of expression and expanding the scale of production

Inactive Publication Date: 2009-03-18
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a ScHAP1 homologue, the function of the Kluyveromyces lactis HAP1 gene (K1HAP1) is currently unknown

Method used

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  • Method for improving expression level of recombinant protein in kluyveromyces
  • Method for improving expression level of recombinant protein in kluyveromyces
  • Method for improving expression level of recombinant protein in kluyveromyces

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of Kluyveromyces chickpea inulinase gene expression plasmid pUKD-S-αF-PinuT

[0034] ①. Construction of Kluyveromyces chickpea inulinase gene expression cassette

[0035] First, using the total DNA of Kluyveromyces chickpea Y179 as a template, and using P1 and P2, P5 and P6 as primers, respectively, the promoter of Kluyveromyces chickpea exoinulinase gene was amplified by PCR technology. The DNA fragment of the subregion, the coding region of the inulinase gene and the DNA fragment of the terminator region, and then using the pPIC9K plasmid as a template and P3 and P4 as primers, the α-Factor signal peptide DNA fragment was amplified by PCR technology. Carry out the corresponding restriction endonuclease treatment according to the restriction enzyme site designed for each fragment, and the three restriction fragments obtained are connected with the plasmid pBS_SK which has undergone double restriction enzyme digestion treatment with EcoR1 and HindIII ...

Embodiment 2

[0048] Example 2 Construction of engineering strain K1-WT / pUKD-S-αF-PinuT and engineering strain klhap1Δ / pUKD-S-αF-PinuT expressing Kluyveromyces lactis gene.

[0049] Plasmid pUKD-S-αF-PinuT was used to transform Kluyveromyces lactis MW179-1D strain and HAP1 gene-deficient strain MW179-1D hap1Δ, respectively, using LiAc transformation method. The sequence of Kluyveromyces lactis HAP1 gene is SEQ1.ID.NO 1.

[0050] ① Inoculate a single colony into 3ml liquid YEPD medium and culture with shaking at 30°C for 16-18 hours.

[0051] ②Dilute the culture into 50ml liquid YEPD to make the initial OD 600 =0.2, culturing with shaking at 30℃ for 3-5 hours, OD 600 = 0.4-0.6.

[0052] ③ Collect the cells by centrifugation at 5K for 5 minutes.

[0053] ④ Discard the culture medium, suspend the cells in 25ml sterile water, and centrifuge as above.

[0054] ⑤ Discard the water, suspend the cells with 1ml of 0.1mol / L LiAc, and transfer to a sterile 1.5ml centrifuge tube.

[0055] ⑥ Pellet the cell...

Embodiment 3

[0060] Example 3 Kluyveromyces lactis engineering bacteria K1-WT / pUKD-S-αF-PinuT and klhap1Δ / pUKD-S-αF-PinuT fermentation culture and inulinase expression analysis

[0061] ①Put K1-WT / pUKD-S-αF-PinuT and klhap1Δ / pUKD-S-αF-PinuT engineering strains of Kluyveromyces lactis into 3ml selective SD liquid medium, respectively, in a shaker at 30℃ and 250rpm After being cultured in medium overnight, it is used as seed solution. The components of the selective SD liquid medium are: 0.67% YNB, 2% glucose, 0.002% uracil and 0.002% methionine.

[0062] ②Put the seed solution into a 150ml Erlenmeyer flask containing 25ml of liquid YEPD medium at a ratio of 1:25, and cultivate for 120 hours in a shaker at 30°C and 250rpm. The components of the liquid YEPD medium are: 1% yeast extract, 2% peptone and 2% glucose.

[0063] ③Aspirate fermentation broth samples every 24 hours, centrifuge at 5000 rpm for 5 min to precipitate the bacteria, and take the supernatant to analyze the expressed product inul...

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Abstract

The invention belongs to the technical field of gene engineering, in particular to a technical proposal used to improve the expression level of recombinant protein. In the method, a promoter, alpha-signal peptide, target protein of an inulase gene are orderly connected with a terminal subcode sequence of the inulase gene first; then the sequence is inserted into a Kluyveromyces lactis expression vector; finally the Kluyveromyces lactis is transformed and fermented, and a fermented supernatant is taken and separated. The method can accelerate the remarkable growth of the yield of the recombinant protein expressed in the Kluyveromyces lactis. The technical proposal can be used to improve the expression amount of various proteins and provide a new method for reducing production cost and enlarging production scale.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a technical scheme for improving the expression level of recombinant proteins. Background technique [0002] Yeast is a single-celled lower eukaryotic organism with the characteristics of both prokaryotic and eukaryotic cells: it is convenient to cultivate, reproduce fast, low cost, easy to gene manipulation, and has a secretory pathway, which can translate eukaryotic gene products. After processing and modification, a correctly folded and active protein is obtained. Therefore, the yeast expression system is known as the most commercially valuable expression system (F.R. Schmidt, Recombinant expression systems in the pharmaceutical industry, Appl Microbiol Biotechnol, 65:363-372, 2004). In addition to the widely used Saccharomyces cerevisiae and Pichia pastoris expression systems, there is also a class of unconventional yeast expression systems represented by Kl...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/81C12N9/24C12N1/19C12R1/645
Inventor 刘建平李育阳俞静袁汉英蔡显鹏
Owner FUDAN UNIV
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