Pichia pastoris wall protein gcw19 and its surface display system and construction method
A construction method and gcw19 technology, applied in the biological field, can solve the problems of low expression of Pir protein and general effect of anchoring protein, and achieve the effect of high expression efficiency
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Embodiment 1
[0037] Example 1: Cloning, expression and identification of Pichia pastoris wall protein GCW19 gene
[0038] (1) Cloning of Pichia pastoris wall protein GCW19 gene
[0039] According to the gene sequence SEQ NO.4 of Pichia pastoris wall protein GCW19 and the characteristics of multiple cloning sites on Pichia pastoris plasmid pPIC9K, synthetic primers were designed:
[0040]
[0041] The box part of primer P1 is EcoR I restriction site, the underlined part is Mlu I restriction site, the italic part is the FLAG tag sequence; the underlined part of primer P2 is notI restriction site. Using Pichia pastoris GS115 genomic DNA as a template and P1 and P2 as primers, the wall protein GCW19 gene sequence was amplified by PCR. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; followed by 30 more cycles : Denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute; finally extension at 72°C for 7 minutes.
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Embodiment 2
[0046] Example 2: Construction of Pichia pastoris wall protein GCW19 surface display vector p9KGCW19
[0047] (1) Cloning of Pichia pastoris wall protein GCW19 gene
[0048] According to the gene sequence of Pichia pastoris wall protein GCW19 and the characteristics of multiple cloning sites on Pichia pastoris plasmid pPIC9K, synthetic primers were designed:
[0049]
[0050] The underlined part of primer P2 is not I enzyme cleavage site; the box part of primer P3 is EcoR I restriction site, the underlined part is Mlu I restriction site. Using Pichia pastoris GS115 genomic DNA as a template and P2 and P3 as primers, the wall protein GCW19 gene sequence was amplified by PCR method. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; followed by 30 cycles or less : Denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute; finally extension at 72°C for 7 minutes. The Pichia pastoris wall protein...
Embodiment 3
[0053] Example 3: Construction of Pichia pastoris wall protein GCW19 surface display vector pZαAGCW19
[0054] (1) Cloning of Pichia pastoris wall protein GCW19 gene
[0055] According to the gene sequence of Pichia pastoris wall protein GCW19 and the characteristics of multiple cloning sites on Pichia pastoris plasmid pPIC9K, synthetic primers were designed:
[0056] P2: 5'-TATTAT GCGGCCGC TCAAATTAACATAGC-3' (SEQ NO: 3)
[0057] P4: 5’ – ATAT CTCGAG GAGCCAATCTTCAC-3' (SEQ NO: 6)
[0058] The underlined part of primer P2 is not I restriction site; the underlined part of primer P4 is xho I restriction site. Using Pichia pastoris GS115 genomic DNA as a template, and P2 and P4 as primers, the wall protein GCW19 gene sequence was amplified by PCR method. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; followed by 30 cycles: 94°C Denaturation for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute; final extension at ...
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