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Preparation method of protein UGTCs4

A protein and recombinant plasmid technology, applied in the biological field, can solve problems such as the inability to obtain high-purity crocetin glycosyltransferase

Inactive Publication Date: 2016-09-21
ECONOMIC CROPS RES INST XINJIANG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But up to now, it is still impossible to obtain high-purity crocetin glycosyltransferase

Method used

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  • Preparation method of protein UGTCs4
  • Preparation method of protein UGTCs4
  • Preparation method of protein UGTCs4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1, the preparation of recombinant crocetin acid glycosyltransferase

[0085] 1. Construction of recombinant plasmids

[0086] 1. The total RNA of the saffron suspension cells was extracted by the Trizol method, and the first-strand cDNA was reverse-transcribed from the total RNA by reverse transcriptase.

[0087] 2. Synthetic primer F1: 5'-C CATATG GAGCAGAAAGATGAGAACGGAA-3' (the underline is the recognition sequence of restriction endonuclease Nde Ⅰ) and R1: 5'-C GTC GAC TTTACAACAATGATCAATGAACTCCT-3' (the underline is the recognition sequence of restriction endonuclease Sal I).

[0088] 3. Using the cDNA obtained in step 1 as a template and F1 and R1 synthesized in step 2 as primers, perform PCR amplification to obtain a PCR amplification product of about 1388 bp. Detected by 1% agarose gel electrophoresis, the results are shown in figure 1 (M is DNA Marker, lane 1 is the PCR amplification product, and the arrow points to the target PCR amplification pr...

Embodiment 2

[0151] Example 2. Using HPLC to detect the glycosyltransferase activity of recombinant crocetin glycosyltransferase under different reaction conditions

[0152] 1. Dialysis

[0153] Under the condition of 4°C, the recombinant crocetin glycosyltransferase solution obtained in Example 1 was dialyzed to desalinate, then replaced into pH 7.5, 5mM Tris-HCl buffer solution, and finally carried out using an ultrafiltration tube with a molecular weight of 30MW. The protein was concentrated by ultrafiltration to obtain a protein sample of the recombinant crocetin glycosyltransferase with a purity of 98%. The protein sample was sequenced for the 25 amino acid residues at the N-terminus, and the results showed that the sequence of the 25 amino acid residues at the N-terminus of the protein was as shown in positions 1 to 25 from the N-terminus of Sequence 4 in the sequence listing.

[0154] 2. Using HPLC to detect the glycosyltransferase activity of recombinant crocetin glycosyltransfera...

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Abstract

The invention discloses a preparation method of protein UGTCs4. The protein UGTCs4 has the croctin acid glycosyl transferase activity. The preparation method comprises the following step of culturing engineering bacteria, so as to obtain the protein UGTCs4, wherein the engineering bacteria is a recombinant bacteria which is obtained by importing a recombinant expression carrier into host bacteria, and the recombinant expression carrier is a recombinant plasmid which is obtained by inserting the expression carrier into an encoding gene of the protein UGTCs4. After proofing by experiments, the preparation method of the protein UGTCs4 has the advantages that the croctin acid can be sequentially glycosylated to form crocin 5 and crocin 3; the important application value is realized for the production of crocin 5 and / or crocin 3, preparation of products with croctin acid glycosyl transferase functions, or catalyzing on glycosylation of croctin acids.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing protein UGTCs4. Background technique [0002] Saffron is also called saffron and saffron, which is the dry stigma of the perennial Iridaceae plant saffron (Crocus sativus L.), and is a kind of famous and precious traditional Chinese medicine in my country. Crocetin is a pigment compound extracted from saffron. Its main components are crocus, crocetin and its derivatives. Crocetin is a series of ester glycosides formed by combining crocetin with different sugars. , is one of the main active ingredients of saffron, has anti-platelet aggregation, anti-thrombosis, anti-myocardial ischemia, anti-cancer and other pharmacological effects, and almost no toxic side effects. Existing studies have shown that crocetin glycosyltransferase has the function of glycosylation crocetin to form crocin. For example, in 2004, Morgan et al. isolated and found UGTCs2 gen...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54
CPCC12N9/1051C12Y204/01271
Inventor 赖成霞王玮石必显李春平刘忠山李金枫
Owner ECONOMIC CROPS RES INST XINJIANG ACAD OF AGRI SCI
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