Coding sequence of Chinese yew tarad-ane C13-lateral chain-N-benzoyl transiting enzymic protein

A technology of benzoyl transferase and coding sequence, which is applied in the fields of genetic engineering and molecular biology, and can solve problems such as no further research.

Inactive Publication Date: 2005-01-05
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In the analysis of the existing literature, although "The Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences Progress) 2002, 99 (14), 9166-9171" reported that taxol was cloned from

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Cloning of taxane C13-side chain-N-benzoyltransferase gene from Taxus mandiae

[0121] 1. Tissue separation (isolation)

[0122] The young leaves of Taxus chinensis (variety "Mandia") were obtained from Southwest Normal University, and the materials were collected and frozen in liquid nitrogen immediately.

[0123] 2. RNA isolation (RNA isolation)

[0124] Take part of the tissue, grind it with a mortar, add it to a 1.5mL EP tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to 1.5mL EP tube, and extract total RNA (TRIzol Reagents, GIBCOBRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0125] 3. Cloning of Full-length cDNA

[0126] According to the amino acid conserved sequences of some plant acyltransferases, conservative primers were designed, and using the principle of homologous gen...

Embodiment 2

[0135] Sequence Information and Homology Analysis of Taxane Taxane C13-Side Chain-N-Benzoyltransferase Gene

[0136] The full-length cDNA of the novel taxoyltransferase of the present invention is 1401bp in length, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at nucleotides 36-1352. According to the full-length cDNA deduced the amino acid sequence of taxoyltransferase, a total of 438 amino acid residues, molecular weight 48410.92, pI 6.10. See SEQ ID NO.4 for the detailed sequence.

[0137] The full-length cDNA sequence of taxane taxane C13-side chain-N-benzoyltransferase and its encoded protein were translated in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundantGenBank CDS by BLAST program Nucleotide and protein homology search in +PDB+SwissProt+Superdate+PIR database, and it was found that it is related to taxane taxane C13-side chain-N-benzoyltransferase (TcDBTNBT) gene (AF466397) It has 78% homology at the nucleotide le...

Embodiment 3

[0139] Prokaryotic expression and purification of Taxus taxane C13-side chain-N-benzoyltransferase in Escherichia coli

[0140] In this example, the full-length taxus TmDBTNBT coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0141] The taxus TmDBTNBT polypeptide was prokaryotically expressed in Escherichia coli in the form of fusion protein.

[0142] Construction of prokaryotic expression vector and transformation of Escherichia coli

[0143] According to the amino acid sequence of Taxus TmDBTNBT, primers for the protein coding region were designed, and restriction endonuclease sites were introduced into the forward and reverse primers (this depends on the pET32a(+) vector selected), so as to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Taxus chinensis TmDBTNBT gene was cloned into the pET3...

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Abstract

The invention T.chinensis taxane C13-side chain-N-benzoyl-transporting proteinase coding sequence, and the separated DNA molecule includes: a nucleotide sequence coding polypeptide with the activity of C13-side chain-N-benzoyl-transporting proteinase, where the nucleotide sequence has at least 70% consanguinity with that (36-1352 nt) in SEQ ID No.3; or the nucleotide sequence can hybridize with that (36-1352 nt) in SEQ ID No.3 at 40-55 deg.C. The invention is an enzyme catalyzing benzoyl CoA and debenzoyl-taxol to generate taxol, and with good help to the health of people.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a taxane C13-side chain-N-benzoyl transferase expressed in Taxus chinensis and its nucleic acid sequence. Background technique [0002] Paclitaxel (taxol) is a natural product with unique anticancer effect extracted from the bark of Taxus brevifolia by Wani et al. in the 1970s. Paclitaxel is currently one of the best natural anti-cancer drugs certified by the FDA. It is the drug of choice for the treatment of ovarian cancer, and it has good curative effects on leukemia, lung cancer, brain cancer and some other solid tumors. Side effects are minimal. Now, the only difficulty facing paclitaxel production is the serious shortage of drug sources. [0003] In recent years, great progress has been made in the research on finding and expanding the source pathway of paclitaxel. Since the biosynthetic metabolic pathway of...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N9/10C12N15/29C12N15/54C12N15/79C12N15/82
Inventor 唐克轩开国银苗志奇李柱刚赵凌侠
Owner SHANGHAI JIAO TONG UNIV
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