Part:BBa_K5070002
pPICZαA-LHyal-WT(op)
pPICZαA-LHyal-WT(op)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 715
- 1000COMPATIBLE WITH RFC[1000]
Construction Design
The original leech hyaluronidase (HAase) possesses a naturally derived coding sequence. We applied codon optimization to mitigate the challenges of heterologous expression in Pichia pastoris. This strategy enhances the expression efficiency of functional leech HAase, thereby improving the yield rate. Furthermore, we used the pPICZαA as an expression vector, which introduced an α-factor secretion signal at the N-terminus to facilitate the secretion of mature leech HAase.
We chose the HAase gene LHyal-WT(op) to produce HAase and used the pPICZαA plasmid as the carrier. This plasmid is common for use in Pichia pastoris (GS115), making it accessible and stable for high school students. We employed PCR and gene sequencing for every preparation step to verify results. Additionally, we used SDS-PAGE and a transparent circle test to assess enzyme activity and productivity, and real-time fluorescence quantitative PCR and DNS to compare the productivity of WT and OP versions of the gene.
Engineering Principle
First, we extracted and digested the pPICZαA plasmid using EcoR1 and Sal1. PCR was then used to amplify the LHyal-WT(op) gene sequence, followed by homologous recombination to link the vector and gene sequence. After successful plasmid construction, heat shock was used to transform E. coli DH5α.
Cultivation, Protein Expression, and SDS-PAGE
We used gel electrophoresis to confirm gene amplification. The LHyal-WT(op) gene, which is 1470 bp, was clearly visible on the gel, indicating successful amplification.
Once the recombinant plasmid was integrated into DH5α, we cultured it for one day, using antibiotics, PCR, and gene sequencing to confirm the correctness of the procedure. The results are shown in Figure 4.
We used enzymes to digest the recombinant plasmid for transformation into yeast. The linearized digests are shown in Figure 5.
We then transformed the recombinant plasmid into yeast and verified its sequence via PCR and gel electrophoresis. We also used antibiotics to ensure selection. The results are shown in Figure 6.
We then used SDS-PAGE to test protein expression, with the HAase protein expressed at around 47 kDa. The enzyme activity was confirmed using a transparent circle test. The SDS-PAGE results are shown in Figure 7.
Characterization/Measurement
HAase activity was measured after induction in both wild-type and optimized yeast strains. The optimized strain exhibited significantly higher HAase activity, suggesting that the optimization strategies enhanced enzyme production. Further analysis quantified these improvements using real-time fluorescence quantitative PCR to compare WT and OP productivity.
Enzyme activity was further measured through a transparent circle test. HAase activity produces glucose upon degrading HA, and the DNS method quantified the glucose produced. A glucose standard curve was sketched to show the expected glucose amount. Yeast was cultivated for 0h, 24h, 48h, 72h, and 96h, and productivity was shown in bar charts, as in Figure 9.
To further conclude the experiment, we used DNS over a 96-hour period to track HAase activity. Initially, the activity of HAase produced by WT was higher than that of OP, but after prolonged induction, the activity of OP exceeded that of WT, as shown in Figure 10.
Summary
The iGEM project "High-Yield Hyaluronidase Pichia pastoris Cell Factory Construction" addresses the challenges associated with the costly and limited production of hyaluronidase (HAase), an enzyme extensively used in medical applications. This project involves genetically modifying Pichia pastoris to produce HAase derived from leeches. Key activities include the design and synthesis of the HAase gene, the creation of expression plasmids, and the transformation of Pichia pastoris with these plasmids. Strains with high-copy integrations are selected through antibiotic resistance.
Enzyme activity is analyzed using SDS-PAGE and DNS methods to optimize production and correlate gene copy number with HAase activity. The ultimate goal is to develop a genetically engineered yeast strain that can produce HAase efficiently, potentially impacting the biopharmaceutical industry by offering a more cost-effective and scalable production method for hyaluronidase.
References
[1] Cui, Y., et al. (2015). High-level expression of human hyaluronidase in Pichia pastoris and its potential application in tumour therapy. Applied Microbiology and Biotechnology, 99(21), 8817-8827.
[2] Liu, C., et al. (2017). Enhanced production of recombinant human hyaluronidase in Pichia pastoris by optimizing codon usage and fermentation conditions. Biotechnology Letters, 39(8), 1233-1239.
[3] Wang, J., et al. (2018). A novel method for quantitative detection of hyaluronidase activity using a DNS assay. Biotechnology Progress, 34(2), 434-440.
[4] Zhang, J., et al. (2019). Metabolic engineering of Pichia pastoris for the production of biopharmaceuticals. Microbial Cell Factories, 18(1), 1-12.
[5] Chen, X., et al. (2020). Synthetic biology approaches to enhance the production of heterologous proteins in yeast. Biotechnology Journal, 15(5), 1-15.
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