A kind of preparation method of high mannose type oligosaccharide linked by phytanol

A high mannose and mannose-based technology, applied in the fields of molecular biology and biochemistry, can solve the problems of difficult expression and purification, and difficult to obtain, and achieve the effect of overcoming high efficiency and stereoselectivity

Active Publication Date: 2021-05-11
JIANGNAN UNIV
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Problems solved by technology

However, no chemical enzymatic method has been reported to synthesize high mannose oligosaccharides. The main reason is that a series of enzymes that catalyze the synthesis of this structure are endoplasmic reticulum resident enzymes, all of which are eukaryotic membrane proteins, and expression and purification are extremely difficult; One reason is that the substrate of the enzyme is a lipid phosphate-linked sugar, which is not easy to obtain

Method used

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  • A kind of preparation method of high mannose type oligosaccharide linked by phytanol
  • A kind of preparation method of high mannose type oligosaccharide linked by phytanol
  • A kind of preparation method of high mannose type oligosaccharide linked by phytanol

Examples

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Embodiment 1

[0028] Example 1: Prokaryotic expression and purification of Saccharomyces cerevisiae Alg1DTM, Trx-Alg2, Alg11DTM and Dpm1

[0029] We used software to analyze the membrane topology of three eukaryotic transmembrane proteins, Alg1, Alg2, and Alg11 (TMHMM Server v. 2.0) ( figure 1A). Alg1 and Alg11 truncated the N-terminal transmembrane structure (Alg1DTM, aa 35-349; Alg11DTM, aa 45-548); Alg2 fused the Trx tag (Trx-Alg2) to construct the following prokaryotic expression vectors: pET28a-Alg1DTM, pET32a- Trx-Alg2 and pET28a-Alg11DTM, and construct vector pET28a-Dpm1. Transform the recombinant prokaryotic expression plasmid into ROSETTA prokaryotic expression host bacteria, spread on LB+ (Kan or Amp) + chloramphenicol (34μg / mL) and pick a single colony from the transformation plate the next day, inoculate in 5mL LB+ (Kan or Amp) + In chloramphenicol liquid medium, culture overnight at 37°C with shaking. Inoculate 2 mL of the overnight cultured bacterial solution into 200 mL of...

Embodiment 2

[0031] Example 2: Prokaryotic expression of Saccharomyces cerevisiae M-Alg3, M-Alg9 and Alg12 and preparation of membrane components

[0032] The membrane topology of three eukaryotic transmembrane proteins of yeast Alg3, Alg9 and Alg12 was analyzed by software (TMHMMServer v. 2.0) ( figure 1 B) Prokaryotic vectors were constructed: pET28a-Alg3, pET28a-Alg9 and pET28a-Alg12, and it was found that both Alg3 and Alg9 were degraded by western blotting ( figure 2 B) Mistic was fused at the N-terminus of Alg3 and Alg9 to construct prokaryotic expression vectors: pET28a-M-Alg3 and pET28a-M-Alg9. First, the corresponding genes were amplified from the Saccharomyces cerevisiae W303A genome and connected to prokaryotic expression Among the vectors, construct recombinant vectors: pET28a-M-Alg3, pET28a-M-Alg9 and pET28a-Alg12. The recombinant prokaryotic expression plasmid was transformed into the ROSETTA prokaryotic expression host strain, and the subsequent induced expression of the p...

Embodiment 3

[0033] Example 3: Enzymatic synthesis of phytanyl-phosphate-Man (PP-Man)

[0034] Standard enzyme reaction conditions are as follows (50 µL system): 50 mM Tris / HCl (pH 7.5), 10 mM MgCl 2 , 1% NP-40, 20 mM phytanyl-phosphate, 50 mM GDP-Man and 2 mg / mL purified Dpm1 protein. Reactions were incubated at 30°C for 10 hours. The reaction efficiency of the enzyme was monitored by TLC.

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Abstract

The invention discloses a method for preparing a phytoalkanol-linked high-mannose oligosaccharide, comprising: prokaryotic expression of mannosyltransferase: respectively expressing yeast-derived Alg1ΔTM, Trx-Alg2, Alg11ΔTM, Dpm1 in Escherichia coli , M-Alg3, M-Alg9 and Alg12; purification of mannosyltransferase; preparation of mannosyltransferase membrane components; synthesis of donor substrate PP-Man; PPGn 2 ‑Man 1 , PPGn 2 ‑Man 3 , PPGn 2 ‑Man 5 , PPGn 2 ‑Man 6 , PPGn 2 ‑Man 7 , PPGn 2 ‑Man 8 , PPGn 2 ‑Man 9 and PPGn 2 ‑Man 4 Synthesis. The present invention expresses and purifies yeast-derived Alg1ΔTM, Trx-Alg2, Alg11ΔTM, Dpm1, M-Alg3, M-Alg9 and Alg12 mannosyltransferase, and successfully prepares PPGn in vitro 2 ‑Man 1 , PPGn 2 ‑Man 3 , PPGn 2 ‑Man 5 , PPGn 2 ‑Man 6 , PPGn 2 ‑Man 7 , PPGn 2 ‑Man 8 , PPGn 2 ‑Man 9 and PPGn 2 ‑Man 4 High mannose-type oligosaccharides solve the technical problems that prokaryotic expression of multiple transmembrane eukaryotic proteins is easy to degrade and the expression and purification of eukaryotic membrane proteins are difficult. Difficulty, promote the development of glycochemistry and glycobiology.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biochemistry, and in particular relates to a preparation method of phytanol-linked high mannose oligosaccharides. Background technique [0002] Glycosylation is the process of modifying proteins or lipids with sugars (oligosaccharides) to form sugar complexes, and it is also one of the main forms of post-translational modification in eukaryotic cells. N-glycosylation modifications on proteins directly affect the structure and function of proteins and have important physiological significance. As a large category of N-glycosylation modifications, high mannose oligosaccharides are currently widely used in biological research, such as glycochips, glycovaccines, and protein quality control. At present, the preparation of high mannose oligosaccharides is mainly through chemical methods and extraction from plants or yeasts. However, both methods are time-consuming and labor-intensive, wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/18C12N15/54C12N15/70C12N9/10
CPCC12N9/1051C12N15/70C12P19/18C12Y204/01083
Inventor 高晓冬李盛陶王宁
Owner JIANGNAN UNIV
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