Kluyveromyces lactis eukaryotic expression method of streptomyces murinus AMP deaminase gene

A technology of Streptomyces griseus and Kluyveromyces, applied in the field of molecular biology, to achieve high and stable enzyme activity

Active Publication Date: 2014-04-30
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now the research on the recombinant expression of AMP deaminase gene in China has never been reported, and the research on the recombinant expression of this enzyme in foreign countries is less, only in the patent "AMP deaminase derived from actinomycetes and Its application" (Applicant: Amano Enzyme Co., Ltd.) reported once

Method used

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  • Kluyveromyces lactis eukaryotic expression method of streptomyces murinus AMP deaminase gene
  • Kluyveromyces lactis eukaryotic expression method of streptomyces murinus AMP deaminase gene
  • Kluyveromyces lactis eukaryotic expression method of streptomyces murinus AMP deaminase gene

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Experimental program
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Embodiment 1

[0026] Cloning of embodiment 1 Streptomyces griseus AMP deaminase gene

[0027] Using the genome of Streptomyces griseis MCCC1A01641 as a template, the primer pair AMPDF3 / AMPDR3 was designed to specifically amplify the AMP deaminase gene of Streptomyces griseris , and XhoI restriction sites and BglII restriction sites were added at both ends of the primer pair. The AMPDF3 / AMPDR3 sequence is as follows: AMPDF3: 5'-GTT CTCGAG GCGCCGCCGCCCCGGCAG-3' (SEQ ID NO: 1); AMPDR3: 5'-GA AGATCT TCACCCCCGGGCGTGCGCCC-3' (SEQ ID NO: 2);

[0028] Purify and recover the PCR product.

Embodiment 2

[0029] Example 2 Construction of recombinant expression vector pKLAC1-AMPD

[0030] The vector pKLAC1 and the PCR product obtained in Example 1 were double-digested with XhoI and BglII respectively, recovered and purified by gel cutting, the digested product was ligated overnight at 16°C, the ligated product was transformed into E.coli JM109, and the pKLAC1-AMPD-JM109 strain plasmid was extracted. The pKLAC1-AMPD-JM109 positive strain was screened out by XhoI and BglII double enzyme digestion. Recombinant expression vector pKLAC1-AMPD enzyme digestion verification electrophoresis picture is as follows figure 1 shown.

Embodiment 3

[0031] Example 3 Electrotransformation of linearized plasmid pKLAC1-AMPD to GG799 and screening of transformants

[0032] Extract the pKLAC1-AMPD-JM109 positive strain plasmid obtained in Example 2, linearize it with BstXI, and electrotransform Kluyveromyces lactis GG799, spread it on a YCB plate, pick a single colony for detection after 3 to 5 days, and extract the recombinant chromosome genome by the helicase method , using specific primers AMPDF3 / AMPDR3 to carry out PCR verification to obtain positive clone transformants; then use the universal primers that come with the pKLAC1 vector to perform PCR to screen out multi-copy transformants. The sequences of the universal primers are shown in Table 1:

[0033] Table 1 General primer sequence of pKLAC1 vector

[0034]

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Abstract

The invention discloses a kluyveromyces lactis eukaryotic expression method of a streptomyces murinus AMP (adenosine monophosphate) deaminase gene. According to the method, a streptomyces murinus genome is used as a template; a specific primer amplification AMP deaminase gene sequence is designed; restriction enzyme cutting sites are respectively introduced to both ends of the AMP deaminase gene; an expression vector pKLAC1 is sub-cloned into the AMP deaminase gene to construct a recombinant plasmid; after enzyme cutting linearization is carried out, kluyveromyces lactis is subjected to electrotransformation; positive clone transformants are screened; then multicopy transformants are screened by utilizing a pKLAC1 universal primer; finally, seed culture and liquid fermentation culture are carried out, fermentation liquor supernatant is taken so as to obtain an AMP deaminase expression product. According to the invention, the AMP deaminase from the streptomyces murinus is successfully expressed in the kluyveromyces lactis for the first time; an experiment result shows that the recombinase has high and stable enzyme activity and can be applied to the fields of foods, medicine and the like.

Description

technical field [0001] The present invention relates to the eukaryotic expression of AMP deaminase gene, in particular to a Kluyveromyces lactis eukaryotic expression method of AMP deaminase gene derived from Streptomyces griseus and the application of the expression product, which belongs to molecular biology technology field. Background technique [0002] AMP deaminase, English name AMP deaminase, abbreviated as AMPD, is an aminohydrolase, which can catalyze the deamination of AMP to generate inosinic acid (IMP) and NH3. IMP is used in food and pharmaceutical fields. important application. In addition, AMP deaminase is one of the three main enzymes in the metabolic cycle of purine nucleotides, which plays an important role in maintaining the body's immunity and adenylate energy charge. Therefore, AMP deaminase is an important enzyme in industrial production. [0003] AMP deaminase widely exists in various organisms. It was first prepared from rat skeletal muscle, and t...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/81C12N9/78
Inventor 张梁石贵阳方炜丁重阳顾正华
Owner JIANGNAN UNIV
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