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Expression and production of therapeutic recombinant human platelet-derived growth factor-BB in Pleurotus eryngii
Jun-Hui Choi1, Seung Kim, Kumar Sapkota
1Department of Biotechnology, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea.
Abstract:
Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is widely used in many therapeutic applications. Until now, there has been no report on rhPDGF-BB expressed in fungi. In this study, we tested whether Pleurotus eryngii could support the expression of human therapeutic rhPDGF-BB protein. A binary vector pCAMBIA1304 containing the hPDGF-BB gene was constructed and introduced into P. eryngii via Agrobacterium tumefaciens-mediated transformation. The transformation of hPDGF-BB gene was confirmed by Southern blot and PCR, whereas the expression was confirmed by Western blot analysis. The recombinant hPDGF-BB reached a maximum expression level of 1.98% of total soluble protein in transgenic mycelia and was in dimeric form. A bioassay revealed that hPDGF-BB expressed in P. eryngii increased proliferation of NIH-3T3 cells similarly to standard material. These results suggest that P. eryngii can be a robust system for the production of human therapeutic proteins including the hPDGF-BB.
Insights
Pleurotus eryngii, a fungus, successfully expressed recombinant human platelet-derived growth factor-BB (rhPDGF-BB). This demonstrates the potential of P. eryngii as a cost-effective system for producing therapeutic proteins.
Area of Science:
- Biotechnology
- Molecular Biology
- Mycology
Background:
- Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is a crucial therapeutic protein.
- Fungal expression systems offer potential advantages for recombinant protein production.
- No prior studies have reported rhPDGF-BB expression in fungi.
Purpose of the Study:
- To investigate the feasibility of using Pleurotus eryngii as a host for expressing human therapeutic rhPDGF-BB.
- To confirm the successful transformation and expression of the hPDGF-BB gene in P. eryngii.
Main Methods:
- Construction of a binary vector (pCAMBIA1304) containing the hPDGF-BB gene.
- Agrobacterium tumefaciens-mediated transformation of P. eryngii with the constructed vector.
- Confirmation of gene integration via Southern blot and PCR.
- Verification of protein expression using Western blot analysis.
- Quantification of rhPDGF-BB expression levels and assessment of its biological activity through cell proliferation assays.
Main Results:
- Successful integration and expression of the hPDGF-BB gene in transgenic P. eryngii mycelia.
- Maximum expression level of rhPDGF-BB reached 1.98% of total soluble protein.
- Expressed rhPDGF-BB was confirmed to be in its functional dimeric form.
- rhPDGF-BB produced in P. eryngii exhibited comparable biological activity to standard material in NIH-3T3 cell proliferation assays.
Conclusions:
- Pleurotus eryngii is a viable and robust host system for the production of recombinant human therapeutic proteins.
- This study establishes a novel fungal platform for rhPDGF-BB production, potentially leading to more economical therapeutic protein manufacturing.
- The findings open avenues for exploring P. eryngii for the expression of other complex human therapeutic proteins.
