Production of Non-N glycosylated protein from yeast

A technology for glycosylated protein and glycoprotein, which is applied in the production field of non-N-glycosylated protein, can solve the problems of loss of enzymatic activity and reduced enzymatic activity, and achieves the effect of simple method and cost reduction.

Inactive Publication Date: 2006-03-15
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the expression of heterologous proteins in the expression system of Pichia pastoris generally has hyperglycosylation phenomenon, and the

Method used

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  • Production of Non-N glycosylated protein from yeast
  • Production of Non-N glycosylated protein from yeast
  • Production of Non-N glycosylated protein from yeast

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Production of non-N-glycosylated erythropoietin (EPO) in Pichia pastoris strains

[0041] 1. Materials: EPO gene: Genbank [gi: 4503588], pPIC9K vector (Invitrogen Ltd), Pichia GS115 strain containing PNGaseF

[0042] 2. Method:

[0043] 1), the construction of surface expression N-glycoamidase yeast strain:

[0044] Using 5-ATATGAATTCGCTCCGCCAGATAATACCGT-3; 5-CACGTCTAGAGTTTGTAACTACCGGAG-3 as primers to clone the N-glycoamidase gene from Pseudomonas meningitidis ATCC 33958 (purchased from ATCC), PCR amplification conditions: 50 μl reaction system, 10×PCR buffer 5 μl , MgCl 2 (25mmol / L) 3μl, dNTP (25mmol / L) 1μl, primers 1 and 2 (20pmol / L) 1μl each, genomic DNA 1μl, Taq DNA polymerase 0.5U, after pre-denaturation at 94°C for 5min, according to the following parameters: 94 Denaturation at ℃ for 45s, annealing at 56℃ for 45s, extension at 72℃ for 2min, 28 cycles, and the last cycle of extension at 72℃ for 10min. The XbaI and NotI sites inserted into the pBluescriptSK(+) ...

Embodiment 2

[0050] Production of non-N-glycosylated interferons in yeast strains

[0051] 1. Materials: Interferon gene: Genbank [gi: 50593016], pPIC9K vector (Invitrogen Ltd), Pichia GS115 strain containing PNGase F

[0052] 2. Method:

[0053] 1), the construction of surface expression N-glycoamidase Pichia strain:

[0054] Using 5-ATATGAATTCGCTCCGCCAGATAATACCGT-3; 5-CACGTCTAGAGTTTGTAACTACCGGAG-3 as primers to clone the N-glycoamidase gene from Pseudomonas meningitidis ATCC 33958 (purchased from ATCC), PCR amplification conditions: 50 μl reaction system, 10×PCR buffer 5 μl , MgCl 2 (25mmol / L) 3μl, dNTP (25mmol / L) 1μl, primers 1 and 2 (20pmol / L) 1μl each, genomic DNA 1μl, Taq DNA polymerase 0.5U, after pre-denaturation at 94°C for 5min, according to the following parameters: 94 Denaturation at ℃ for 45s, annealing at 56℃ for 45s, extension at 72℃ for 2min, 28 cycles, and the last cycle of extension at 72℃ for 10min. The XbaI and NotI sites inserted into the pBluescriptSK(-) vector af...

Embodiment 3

[0059] Production of non-N-glycosylated ribonuclease B in yeast strains

[0060] 1. Materials: ribonuclease B gene, pPIC9K vector (Invitrogen Ltd), Pichia GS115 strain containing PNGase F

[0061] 2. Method:

[0062] 1), the construction of surface expression N-glycoamidase Pichia strain:

[0063] Using 5-ATATGAATTCGCTCCGCCAGATAATACCGT-3; 5-CACGTCTAGAGTTTGTAACTACCGGAG-3 as primers to clone the N-glycoamidase gene from Pseudomonas meningitidis ATCC 33958 (purchased from ATCC), PCR amplification conditions: 50 μl reaction system, 10×PCR buffer 5 μl , MgCl 2 (25mmol / L) 3μl, d NTP (25mmol / L) 1μl, primers 1 and 2 (20pmol / L) 1μl each, genomic DNA 1μl, Taq DNA polymerase 0.5U, pre-denaturation at 94°C for 5min, according to the following parameters: Denaturation at 94°C for 45 s, annealing at 56°C for 45 s, extension at 72°C for 2 min, 28 cycles, and the last cycle of extension at 72°C for 10 min. The XbaI and NotI sites inserted into the pET22b vector after digestion with XbaI a...

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Abstract

Production of non-glycosylated protein from yeast is carried out by constructing expression carrier containing N-glycoamidase and purposive protein gene from molecular biological technology, transferred into yeasts from electric transformation, expressing N- glycoamidase on yeast cell surface, excretory expressing purposive protein into culture medium, removing chain of glucoprotein excretory expressed from N- glycoamidase surface expressed and obtaining non-glycosylated protein. It has better expression protein activity and correct tertiary structure and no immunogenicity to human body.

Description

technical field [0001] The invention relates to a method for producing non-N-glycosylated protein, in particular to a method for producing non-N-glycosylated protein by using yeast. Background technique [0002] N-glycosamidase (Peptide-N-(N-acetyl-β-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) is mainly found in Gram-negative bacteria Pseudomonas meningosepticum (Flavobacterium meningosepticum), Saccharomyces cerevisiae and some mammals In the cell, it can specifically break the amide bond between the sugar chain and the protein, generate a complete sugar chain and protein, and convert the amide on the protein into aspartic acid. Because N-glycoamidase can Completely excise N-linked oligosaccharides (N-glycosides) from glycopeptides and glycoproteins, and completely separate proteins and sugar chains, which is useful for the analysis of cell surface sugar structure-function and the function of sugar chains on glycoproteins The analysis and research of the impact ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/81C12N15/52C12P21/02
Inventor 祁庆生苏移山王鹏
Owner SHANDONG UNIV
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