Related biological materials of fusion protein Nt4CL3aPcSTS and application of related biological materials

A fusion protein and biomaterial technology, applied in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problem of high-level accumulation of resveratrol

Active Publication Date: 2015-05-13
BEIJING UNIV OF AGRI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] There are many rate-limiting steps in the biosynthetic pathway of resveratrol in organisms. Although the simple overexpression of a certain key enzyme may have a certain promotion effect on the content of resveratrol, due to the limitations of other steps, the high concentration of resveratrol Level accumulation is very difficult

Method used

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  • Related biological materials of fusion protein Nt4CL3aPcSTS and application of related biological materials
  • Related biological materials of fusion protein Nt4CL3aPcSTS and application of related biological materials
  • Related biological materials of fusion protein Nt4CL3aPcSTS and application of related biological materials

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1. Prokaryotic expression and purification of the fusion protein Nt4CL3aPcSTS

[0113] 1. Construction of Nt4CL3aPcSTS gene expression plasmid

[0114] The DNA molecule (Nt4CL3aPcSTS gene) shown in SEQ ID No.2 is artificially synthesized, which encodes the fusion protein Nt4CL3aPcSTS shown in SEQ ID No.1.

[0115] Replace the DNA between the EcoR I recognition sequence and Xho I recognition sequence of pET30a(+) (product of Novagen) with a DNA molecule whose nucleotide sequence is SEQ ID No. 2, and keep the other sequences of pET30a(+) unchanged. Nt4CL3aPcSTS gene expression vector, its name is pET30a-Nt4CL3aPcSTS. pET30a-Nt4CL3aPcSTS expresses the fusion protein Nt4CL3aPcSTS shown in SEQ ID No. 1.

[0116] Replace the DNA between the EcoR I recognition sequence of pET30a(+) and the Xho I recognition sequence with the DNA molecule whose nucleotide sequence is 1636-2802 in SEQ ID No. 2. Keep the other sequences of pET30a(+) unchanged Obtain the PcSTS gene expression ve...

Embodiment 2

[0127] Example 2. HPLC detection of fusion protein Nt4CL3aPcSTS in vitro enzymatic reaction and product

[0128] 2.1. Resveratrol standard product HPLC detection: accurately weigh 1.0 mg of resveratrol standard product (product of Sigma) into a 10 mL volumetric flask, dissolve it with chromatographic methanol, dilute to 10 mL, and configure it to 100 μg / mL white Mother liquor of Veratrol. It was filtered through a 0.22μm filter membrane and diluted with methanol to obtain standard solutions with concentrations of 2.5, 5, 12.5, 25, and 50μg / mL respectively. The concentration was injected from low to high. Use with Sunfire TM Waters HPLC analysis on a C18 reverse phase column (5.0 μm, 4.6 mm×250 mm). The mobile phase is composed of methanol (A) and water (B), and the flow rate is 0.8 mL / min. The following gradient elution conditions are used: within 0-30 min, the volume percentage of methanol in the mobile phase increases from 10% to 70 at a constant rate %, the volume percent...

Embodiment 3

[0135] Example 3. Production of resveratrol by recombinant E. coli

[0136] 3.1 Recombinant Escherichia coli Rosetta / pET30a-Nt4CL3aPcSTS to produce resveratrol: Inoculate the recombinant Escherichia coli Rosetta / pET30a-Nt4CL3aPcSTS in Example 1 in LB liquid medium, and shake culture overnight at 37°C to obtain culture broth 1. The culture broth 1 was inoculated at 1:100 (volume ratio) into an Erlenmeyer flask containing 100mL LB liquid medium (containing 50μg / mL kanamycin and 25μg / mL chloramphenicol), and shake culture to OD 600 Value between 0.4 and 0.5 to obtain culture broth 2, add IPTG to culture broth 2, call the resulting system the culture broth 2 system, and make the concentration of IPTG in the culture broth 2 system 1.0 mM, Induce culture at 25℃, 150rpm for 4h, centrifuge at 6000rpm for 2min, discard the supernatant, and use 100mL medium containing 50μg / mL kanamycin and 25μg / mL chloramphenicol (this contains 50μg / mL kanamycin and 25μg / mL The chloramphenicol medium is a ...

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Abstract

The invention discloses related biological materials of fusion protein Nt4CL3aPcSTS and application of the related biological materials. The related biological materials of the fusion protein Nt4CL3aPcSTS provided by the invention comprise the fusion protein Nt4CL3aPcSTS, wherein the fusion protein Nt4CL3aPcSTS is obtained by connecting protein Nt4CL and protein PcSTS through three amino acid residues, and the protein Nt4CL is a protein constituted by 1st-542nd amino acid residues from the Nth end of amino acid sequence SEQ ID No.1; the protein PcSTS is a protein showed by the 546th-933rd amino acid residues from the Nth end of amino acid sequence SEQ ID No.1. The related biological materials of the fusion protein Nt4CL7aPcSTS can be used for catalyzing 4-cumaric acid and malonyl coenzyme A to generate resveratrol with bioactivity.

Description

Technical field [0001] The present invention relates to related biological materials and applications of the fusion protein Nt4CL3aPcSTS in the field of biotechnology. Background technique [0002] Resveratrol (Res for short), its chemical name is 3,4',5-trihydroxy-1,2-stilbene (trans-3,4',5-trihydroxystilbene), also known as stilbene, It is a non-flavonoid polyphenol compound containing stilbene structure with a molecular weight of 228.2, insoluble in water, soluble in organic solvents such as ethanol. Natural resveratrol is found in many plants, such as knotweed, peanuts and grapes. [0003] Resveratrol is a plant hormone that increases sharply when plants are exposed to ultraviolet radiation, mechanical damage and fungal infection. Studies have shown that resveratrol has anti-cancer, cardiovascular protection, anti-oxidation, anti-aging, immune regulation, neuroprotection, and The effects of phytoestrogens can be widely used in medicine, health care products, food, cosmetics a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N9/10C12N9/00C12N15/70C12N1/21C12N15/81C12N1/19C12P7/22
Inventor 郭辉力马兰青李相阳吕鹤书相薇马雅迪候羽罗在柒杨亚东刘文彬张红冯静刘欢贾晓堃张振宇
Owner BEIJING UNIV OF AGRI
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