Novel expression enzyme yeast gene engineering system

A technology of genetically engineered bacteria and genes, applied in the field of yeast genetic engineering systems, can solve the problems of no product development, no work report, etc.

Inactive Publication Date: 2008-04-09
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the 1980s, a genetic engineering system has also been built, and many research papers have reported the expression of some exogenous genes [Fleer R., Yeh P. Amellal N., 1991, Stable multicopy vectors for high level secretion of recombinant human serum malbumin by Kluyveromyces yeast, Biotechnology, 9(10): 968-975, Chen Xinjie, Gao Buyu, Shi Wenger et al., 1992, Expression and secretion of human αA interferon in Kluyveromyces lactis, Acta Genetics, 19(3) : 284-288], but it

Method used

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  • Novel expression enzyme yeast gene engineering system
  • Novel expression enzyme yeast gene engineering system
  • Novel expression enzyme yeast gene engineering system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of Kluyveromyces chickpea inulinase gene expression cassette

[0049] First, using the total DNA of Kluyveromyces chickpea Y179ura3- as a template, using P1 and P2, P3 and P4, and P5 and P6 as primers, the promoter and terminator of the inulinase gene were amplified by PCR and the DNA fragment sequenced with the inulinase gene sequence with the signal peptide sequence, and the corresponding restriction endonuclease treatment was carried out according to the enzyme cutting site designed for each fragment, and then these three fragments were combined with the plasmid pRS426 digested by Sal1 -L four factors were connected to obtain plasmid 426-L-PInuT, which included the promoter of the inulinase gene, the terminator and the Sal1 fragment sequenced by the inulinase gene with the signal peptide sequence, which was spliced Inulinase gene expression cassette. Among them, pRS426-L is derived from pRS426, and the structure of pRS426 can be found in: Siko...

Embodiment 2

[0065] Example 2 Construction of Kluyveromyces chickpea inulinase gene expression plasmid pUKD-S-PInuT

[0066] First, the plasmid 426-L-PIT-T in Example 1 was excised with the restriction endonuclease Sall to cut out the DNA fragment with the inulinase gene expression cassette. After the fragments were separated, the sticky ends were filled in with Klenow enzyme; the Kluyveromyces chickpea vector pUKD-S was cut with restriction endonuclease Sse3871, and the sticky ends were also filled in with Klenow enzyme. Then, the two are connected with DNA ligase to obtain the Kluyveromyces chickpea inulinase gene expression plasmid pUKD-S-PInuT.

Embodiment 3

[0067] Example 3 Inulinase Gene Expression Plasmid pUKD-S-PInuT Using LiAc Complete Cell Transformation Method

[0068] ① Inoculate a single colony into 3ml YEPD and culture overnight at 30°C with shaking.

[0069] ② Dilute the overnight culture into 50ml YEPD (10 tubes can be made) so that the initial concentration is O.D 600 =0.08, shake culture at 30℃ for 4.5h to O.D 600 = 0.32. (Initial concentration and incubation time have a great influence on the transformation efficiency.)

[0070] ③5K, 5min centrifugation to collect bacteria

[0071] ④ Discard the culture medium, add sterile water at 5K, and collect the bacteria by centrifugation for 5 minutes.

[0072] ⑤ Wash once more with sterile water, centrifuge at 5K for 5 minutes to collect bacteria.

[0073]⑥ Add 10ml of 0.1M LiAC, incubate in a 30°C water bath for 10min, and divide the cells into 1.5ml centrifuge tubes, 1ml per tube.

[0074] ⑦Centrifuge the cells at high speed and discard the supernatant.

[0075] ⑧ A...

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Abstract

The invention pertains to the biological gene engineering technical field, in particular to a yeast gene engineering system of expressing enzyme which comprises the construction of an expression box which consists of Bengal grain kluyveromyces promoter, terminator and inulase gene with signal peptide, the construction of high-stable inulase gene expression plasmid by inserting the expression box into a carrier pUKD-S, the construction of gene engineering bacteria of expressing inulase with high efficiency by importing Bengal grain kluyveromyces host Y179ura3- and the production of inulase by using the constructed Bengal grain kluyveromyces gene engineering bacteria. The invention could also be used for expressing and producing other endogenetic, exogenous, natural and recombinant enzymes.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to a yeast genetic engineering system for expressing enzymes. Background technique [0002] Yeast system is an important expression system for genetic engineering. Since Hitzman first expressed human alpha interferon with Saccharomyces cerevisiae in 1981, a variety of yeasts have built genetic engineering expression systems, including: Schizosaccharomyces pombe, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Kluyveromyces lactis, Candida utilitis, Schwanniomyces occidenalis, etc. The most widely used of these yeast expression systems are Saccharomyces cerevisiae and Pichia pastoris systems. [0003] Saccharomyces cerevisiae has long been used as a food microorganism in the production of bread and beer, so it is a recognized safe microorganism; secondly, Saccharomyces cerevisiae is also widely used in basic research as a model organis...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12N1/19
Inventor 李育阳袁汉英蔡显鹏张晋文铁桥
Owner FUDAN UNIV
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