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Site directed mutagenesis thermoplasma acidophilum F3 factor recombinant protein and application thereof

A recombinant protein and site-directed mutagenesis technology, applied in the biological field, can solve the problems of increased drug screening cost, high production cost of aminopeptidase, poor stability of aminopeptidase, etc., and achieves high yield, simple preparation method and good thermal stability. Effect

Inactive Publication Date: 2011-10-12
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its expression is expressed in eukaryotic cells, so commercial aminopeptidase has the disadvantages of high production cost and low yield
In addition, commercialized aminopeptidases have poor stability and are not suitable for long-term storage, which also increases the cost of drug screening

Method used

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  • Site directed mutagenesis thermoplasma acidophilum F3 factor recombinant protein and application thereof
  • Site directed mutagenesis thermoplasma acidophilum F3 factor recombinant protein and application thereof
  • Site directed mutagenesis thermoplasma acidophilum F3 factor recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Cloning obtains the mutant gene of Thermoplasma acidophilum F3 factor

[0032] Primers were designed according to the full-length nucleotide sequence of Thermoplasma acidophilum F3 factor published on NCBI, as shown in the following table:

[0033]

[0034] Use the designed pair of primers for PCR amplification, and the reaction system is as follows:

[0035] reaction system:

[0036]

[0037] The PCR reaction conditions are as follows:

[0038] ① Pre-denaturation: 95°C for 5 minutes

[0039] ② Denaturation: 94°C 30s

[0040] ③Annealing: 54℃ 30s

[0041] ④Extension: 72℃ for 2min

[0042] ②-④ cycle 28 times

[0043] ⑤ Final extension: 72°C 10min

[0044] PCR product recovery

[0045] After the PCR, the length of the fragment was analyzed by 1% agarose gel electrophoresis, and the target band was cut out according to the size of the fragment, and the gel cutting product was recovered using the DNA purification kit of Biotech.

Embodiment 2

[0046]Example 2: Transforming the mutant gene into an expression host to obtain a positive expression strain.

[0047] Double digestion reaction of PCR product and plasmid vector

[0048] Enzyme digestion system for PCR products:

[0049]

[0050]

[0051] Reaction conditions: react at 37°C for 2-3h.

[0052] Enzyme digestion system for plasmid vectors:

[0053]

[0054] Reaction conditions: react at 37°C for 6-8h.

[0055] The PCR product and the vector digested product were subjected to 1% agarose gel electrophoresis, and were purified and recovered using a DNA gel recovery kit.

[0056] Connection reaction system:

[0057]

[0058] Mix well and centrifuge for a few seconds, collect the drop on the tube wall to the bottom of the tube, and connect overnight at 16°C.

[0059] Transformation of recombinant plasmids

[0060] (1) Preparation of Competent Cells

[0061] ①Pick a single colony of BL21 (or pick a preserved strain) and inoculate it into about 10ml o...

Embodiment 3

[0083] Example 3: Fermentation and culture of positive expression strains, isolation and purification of point-mutated thermophilic F3 factor recombinant proteins

[0084] Seed culture: Pick positive clones in a conventional method and place them in 5 mL of liquid LB medium containing corresponding antibiotics, and shake and culture at 37°C for 5-6 hours;

[0085] Bacteria expansion culture: Inoculate the seeds into 1L of liquid medium containing the corresponding antibiotics, culture with shaking at 37°C until the bacterial concentration OD600 is 0.8, then cool down to 15°C; add IPTG with a final concentration of 0.6mM after 1 hour, and induce overnight Express.

[0086] Collect the bacteria: 4200rpm, centrifuge at 4°C for 15min, discard the supernatant, and harvest the bacteria; add resuspension solution (25mM Tris-HCl, pH8.0, 100mM NaCl), shake and precipitate the bacteria cells; add protease inhibitor PMSF ( Phenylmethyl sulfonyl fluoride, benzoic acid sulfonyl fluoride) ...

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Abstract

The invention discloses site directed mutagenesis thermoplasma acidophilum F3 factor recombinant protein and application thereof. The amino acid sequence of the recombinant protein is shown as SEQ ID NO.2. The recombinant protein is prepared by the following steps of: connecting full-length segments of thermoplasma acidophilum F3 factor recombinant protein genes subjected to site directed mutagenesis (E101Q, N261T) to a modified expression vector pGL01; transforming escherichia coli BL21 (DE3) by a thermal shock method; inducing to efficiently express by using isopropyl thiogalactoside; and separating and purifying. The recombinant protein has protein active sites consistent with human aminopeptidase active sites, high enzyme activity and high thermal stability, can be used for evaluating the inhibition effect of the newly synthesized aminopeptidase inhibitor, and can replace commercial APN to screen inhibition activity of synthetic compounds.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a recombinant protein of F3 factor of Thermoplasma acidophilum with active site mutation and its application in antitumor drug screening. Background technique [0002] Aminopeptidase N (APN, CD13, EC3.4.11.2) is a type II membrane-bound glycoprotein consisting of 976 amino acids with a molecular weight of about 150KD. It belongs to the Gluzincins of the zinc ion-dependent metalloprotease and aminopeptidase M1 family The subfamily exists in the cell membrane in the form of homodimers. Tumor cell biology studies have shown that aminopeptidase N (Aminopeptidas N, APN / CD13) is closely related to the occurrence and development of invasion and metastasis of malignant tumors. Therefore, research on targeted anticancer drugs targeting APN has attracted much attention. The research on natural products and synthetic inhibitors of small molecule APN inhibitors is relatively mature. Among them...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C12Q1/37C12R1/01
Inventor 谷立川许素娟苏静
Owner SHANDONG UNIV
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