Endogenous signal peptides of Pichia pastoris and application thereof
A Pichia pastoris, signal peptide technology, applied in the biological field, can solve the problem of not very ideal protein secretion effect and so on
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Embodiment 1
[0148] Example 1, β-galactosidase is the guided expression and secretion effect detection of the signal peptide of the reporter gene in Pichia pastoris
[0149] The correct plasmids pG1MFL, pG1DSEL, pG1GASL, pG1DANL, pG1MSBL and pG1FREL verified by sequencing were linearized with restriction endonuclease Sal I, respectively. After purification, the digested products were electrotransformed into pre-prepared Pichia GS115 competent cells. Positive transformants were screened on the MD plate without organic nitrogen source, and PCR was carried out ( Figure 10 ) and sequencing verification, the corresponding recombinant strains were obtained.
[0150] The signal peptide of the constructed β-galactosidase reporter gene was expressed in Pichia pastoris, and the intracellular and extracellular enzyme activities of each β-gal expression strain containing different signal peptides were investigated using YPD as the medium. Depend on Figure 11 It can be seen that the intracellular ...
Embodiment 2
[0156] Example 2, EGFP expression and secretion effect detection in Pichia pastoris
[0157] The constructed plasmids pG1MSBg, pG1FREg or pG1MFg were linearized with restriction enzyme Sal I respectively, after purification, they were transformed into Pichia pastoris GS115 competent cells by electroporation to integrate into the genome. Utilizing the histidine-deficient characteristics of the host strain GS115, the recombinant bacteria were screened on the MD basic medium plate without organic nitrogen source, and verified by PCR ( Figure 12 ), to obtain the corresponding recombinant bacteria G / MSBg, G / FREg and G / MFg.
[0158] The signal peptide of the constructed EGFP reporter gene was expressed in Pichia pastoris, and single colonies of EGFP expressing strains containing different signal peptides were picked and shaken overnight at 30°C and 200rpm in a 2mL YPD test tube. Draw 500 μL of the bacterial solution in the YPD test tube and culture it in a 250 mL shake flask conta...
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