Pichia pastoris for expressing foreign proteins, construction method of pichia pastoris and induced expression method of pichia pastoris
A Pichia pastoris and exogenous protein technology, applied in the field of Pichia pastoris, can solve the problem of low expression of AOX1 and achieve the effect of high cell concentration and high EGFP content
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0047] Example 1 Construction method of ΔGTP1 strain
[0048] The test materials used and their sources include:
[0049] DNA Marker, protein Marker, restriction enzymes (EcoRI, BamHI, HindIII, XbaI, KpnI, AvrII), T4 DNA ligase, and LA Taq DNA polymerase were all purchased from TaKaRa Company.
[0050] Plasmid extraction kits, gel recovery kits, PCR product column purification kits, protein quantification kits, kanamycin, bleomycin, and G418 antibiotics were purchased from Shanghai Sangong Bioengineering Company.
[0051] Primers were synthesized by Shanghai Sangon Bioengineering Company; yeast extract, peptone (OXOID, UK), and other reagents were domestic analytical grade.
[0052] DNA electrophoresis instrument, gel imager, protein electrophoresis instrument (Beijing Liuyi Instrument), PCR instrument (Hangzhou Longji Scientific Instrument Co., Ltd.), spectrophotometer (Shanghai Minorda Instrument Co., Ltd.), ultrasonic breaker (Ningbo New Zhi Biotechnology Co., Ltd.), micr...
Embodiment 2
[0073] Example 2 Determination of AOX1 enzyme activity
[0074] The ΔGTP1 strain and wild-type X-33 stored on the YPD plate were inoculated in the YPD liquid medium for activation, and all the bacteria were collected by centrifugation in the logarithmic phase, and resuspended in a solution containing 0.5% methanol, 0.5% glycerol, 0.25% glycerol, 0.5% glycerol and 0.5% methanol, 0.25% glycerol and 0.5% methanol as carbon source YNB liquid medium, after 48 hours of culture, the protein was extracted, and the AOX1 enzyme activity was determined after protein quantification by Bradford method.
[0075] The determination of AOX1 enzyme activity is as follows: add 100uL of 100μL alcohol oxidase detection solution to the microplate well, then add 5μL of extracted protein, add 3μL of methanol to start the reaction, react at room temperature for 15-30 minutes, until the color develops, add 20μL 2M sulfuric acid (containing 0.1M sodium sulfite) terminated the reaction, and measured the ...
Embodiment 3
[0078] Example 3 Induced expression of foreign protein
[0079] 3.1 Construction of EGFP expression vector
[0080] With EGFP as exogenous protein, such as Picture 1-1 , Figure 1-2 As shown, the EGFP coding sequence was inserted downstream of the AOX1 promoter and GAP promoter of the vectors pPICZB and pPGAPZB to obtain EGFP expression vectors, which were PAOX1-EGFP and Pgap-EGFP, respectively.
[0081] The specific operation is as follows: use EcoRI and XhoI to transform the plasmid pMD TM 19-T-EGFP, pPICZB, Pgapzb double digestion; pMD TM The 19-T-EGFP digestion product EGFP was recovered and purified with a gel recovery kit, and the yeast expression vector pPICZB and Pgapzb digestion products were recovered with a PCR purification kit; T4 ligase was used to connect the fragment and plasmid to obtain the EGFP expression vector Paox1-EGFP respectively and Pgap-EGFP, transformed into competent E.coli DH5α, and then screened with LLB plates containing bleomycin resistance....
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More