Production of Non-N glycosylated protein from yeast

[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(+) ...

[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...

[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...