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Recombinant human fucosyltransferase variant and application thereof

A technology of base transfer and mutation, applied in the field of protein mutants, can solve problems such as unsatisfactory clinical treatment, reduced enzyme activity, and poor enzyme activity

Pending Publication Date: 2022-04-19
北京睿脉医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, neither bacterial nor human fucosyltransferases have good enzymatic activity on macromolecular donor substrates.
The inventors found in the research that, compared with the small molecule GDP-Fucose, the enzymatic activity of fucosyltransferase on the macromolecule donor substrate is nearly a thousand times lower, which cannot meet the needs of clinical treatment

Method used

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  • Recombinant human fucosyltransferase variant and application thereof
  • Recombinant human fucosyltransferase variant and application thereof
  • Recombinant human fucosyltransferase variant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1. Preparation of recombinant fucosyltransferase

[0030] 1.1 Plasmid preparation

[0031] Entrust Suzhou Junji Biotechnology Co., Ltd. to synthesize plasmid DNA and clone it into the vector pRM293 (pRM293 was obtained by transformation on the basis of plasmid pTT5, see Shi C. Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells. Biochemistry. 2005;44(48):15705–15714.), respectively used to express FUT6, FUT10, FUT11 recombinant proteins (amino acid sequences were found in GenBank: M98825.1 or CCDS12152.1, or The SEQ ID NO.1 of this application; GenBank: AJ431184.1 or CCDS6088.1 and GenBank: BC036037.1 or CCDS7333.1, were constructed to express plasmids FUT6-pRM293, FUT10-pRM293, FUT11-pRM293. For specific operation methods, refer to " "Molecular Cloning Experiment Guide", the plasmid was first transformed into DH10B, sequenced, bacteria preserved and cultured ...

Embodiment 2

[0053] Example 2. Activity detection of recombinant fucosyltransferase

[0054] Purified FUT6, FUT10, FUT11 recombinases were analyzed and measured for enzymatic activity. The reaction buffer contained 40ng of recombinases and 100 µM ultrapure GDP-fucose (GDP-Fucose) (Promega Cat.#VA1097) as a donor substrate, and 40 µM fetuin (Promega Cat#V4961) as the acceptor substrate. All enzymatic reactions were performed in white 96-well plates in 25 µL volumes. Incubate for 60 minutes at room temperature. The GDP glycosyltransferase assay was performed as described in the manual (Promega GDP-Glo™ Glycosyltransferase Assay).

[0055] Contains 40 ng of recombinant fucosyltransferase and 100 µM GDP-fucose as donor substrate and double dilution (40 µM) of fetuin as acceptor substrate in a 25 µl reaction volume of PBS buffer and incubate at 37°C for 30 minutes. Then add 25 µl GDP Detection Reagent at room temperature, incubate for 60 minutes, put into GloMax® 96 microplate luminescence ...

Embodiment 3

[0062] Example 3. Preparation and activity detection of mutant FUT6 recombinase

[0063] There are currently no studies showing how fucosyltransferases recognize donor substrates, nor data for structural elucidation. According to the above experiments, it is confirmed that FUT6 can recognize not only small molecule donor substrates, but also macromolecular donor substrates. To improve the ability of FUT6 to recognize macromolecular donor substrates, the amino acid sequence analysis of FUT6 was performed. There are 11 human-derived fucosyltransferases, which belong to the Glycosyltransferse family 10 (Glycosyltransferse family 10) with the prokaryotic fucosyltransferases, but there is no obvious homology in the amino acid primary sequence. Sequence comparison between FUT6 and the alfa-1,3 fucosyltransferase of Helicobacter pylori revealed that FUT6 has 7 "gap" (Gap), that is, in the wild-type amino acid sequence shown in SEQID NO. 1 Amino acid 109 to amino acid 110, amino aci...

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Abstract

The invention discloses a group of recombinant human fucosyl transferase variants, which are used for changing the transfer activity of fucosyl by replacing, increasing, decreasing or deleting specific sites and fragments, so that donor molecules containing GDP-Fucose with the transfer molecular weight of more than 100KD are remarkably improved. Under the action of the enzyme mutant, donor molecules with large molecular weight (such as GDP-Fucose coupled with antibody molecules or recombinant proteins) can be transferred to receptors GlcNAc (N-acetylglucosamine) and LacNac (N-acetyllactosamine). Receptors can be present on the surfaces of macromolecular substances and cell membranes. The invention also discloses an application of the fucosyltransferase mutant in molecular markers and living cell markers.

Description

technical field [0001] The invention relates to a protein mutant and belongs to the technical field of polypeptides. Background technique [0002] Glycosylation is the process by which carbohydrates are covalently attached to target molecules (usually proteins and lipids). Glycosylation of protein molecules is one of the most abundant post-translational modifications. This modification has multiple functions, such as participating in The correct folding of protein molecules regulates the thermodynamic and dynamic stability of proteins, participates in intermolecular interactions and intercellular adhesion, and participates in immune recognition or immune escape. Unlike DNA transcription or protein translation, the glycosylation process of proteins has no template and is an enzymatic reaction. The donor molecule is usually an activated nucleotide sugar. Specific glycoconjugate reactions at the body site (hydroxyl or other functional group). As a component of sugar chains in...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/85C12N5/10C12N5/00A61K39/395A61P35/00A61P29/00A61P3/00
CPCC12N9/1051C12N15/85C12N5/0006A61K39/395A61P35/00A61P29/00A61P3/00C12Y204/01065C12N2800/107
Inventor 王冀姝黄滔
Owner 北京睿脉医药科技有限公司
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