Gene for coding protease K and strain for expressing protease K
A protease and bacterial strain technology, applied in the field of genetic engineering, can solve the problems of low expression of production strains and inability to meet production needs
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Embodiment 1
[0072] Example 1: Codon optimization of the gene encoding proteinase K
[0073] Wild-type proteinase K, derived from Candida albicans Limbersii, GenBank: X14689.1, full-length gene 1155bp, including signal peptide (1-45bp), leader peptide (46-315bp) and mature region (316-1155bp) three part. The fragment without signal peptide is 1110bp (46-1155bp) in length, as shown in SEQ ID NO.1, and the GC content is 58.65%; the wild-type codon is optimized to obtain two optimized proteinase K gene sequences, as shown in Optimized sequence 1 and optimized sequence 2 shown in SEQ ID NO.2 and SEQ ID NO.3. The GC content and CAI index of optimized sequence 1 were 46.85% and 0.91, respectively; the GC content and CAI index of optimized sequence 2 were 44.53% and 0.82, respectively.
Embodiment 2
[0074] Example 2: Construction of single-copy recombinant proteinase K strain
[0075] The wild-type proteinase K and the two codon-optimized sequences were artificially synthesized by BGI, respectively. The restriction sites used were EcoRI and NotI, and the vectors were named pPICZαA-ProK, pPICZαA-ProK1 and pPICZαA-ProK2, respectively. (all contain leader peptide), that is, a single-copy proteinase K expression plasmid. The expression cassette specifically includes: AOX1 promoter, α signal peptide, proteinase K gene, AOX1 terminator, four expression elements.
[0076] Plate-activated cloned strains E.coli DH5α / pPICZαA-ProK, E.coli DH5α / pPICZαA-ProK1 and E.coli DH5α / pPICZαA-ProK2 were inoculated into LB containing 25 μg / mL zeocin, respectively. The liquid medium was cultured at 37°C overnight, the recombinant plasmids were extracted respectively, and the concentration and purity of the plasmids were detected by Nanodrop. Take 5-10 μg of the plasmid, which was linearized wit...
Embodiment 3
[0085] Example 3: Inducible expression of single-copy recombinant proteinase K strains
[0086] The positive transformants corresponding to the wild type, optimized sequence 1 and optimized sequence 2 obtained in Example 2 were selected (2# transformants were selected for the optimized sequence), and single clones were picked and inoculated in 10 mL YPD liquid medium, 30 ° C, 200 rpm Shake culture for 16h (overnight culture), transfer to 50mL BMGY medium at 2% inoculum the next day, and culture to OD at 30°C and 200rpm 600 reach 2 to 6. The culture solution was centrifuged at 5000rpm for 5min at room temperature, and the obtained cells were resuspended in BMMY medium to make the OD 600 ≈ 1.0, continue shaking culture under the same conditions, take samples every 24h, and add methanol to the medium to a final concentration of 0.5% (V / V) to induce proteinase K expression. The samples taken at each time point were centrifuged and filtered, stored at -20°C, and samples with the ...
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