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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

AI Technical Summary

Problems solved by technology

In addition, the expression of heterologous proteins in the expression system of Pichia pastoris generally has hyperglycosylation phenomenon, and the high degree of glycosylation of proteins can reduce the enzyme activity of some foreign proteins (such as: enzymes), or even lose the enzyme activity.

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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