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큰느타리버섯에서 석충 페리틴 단백질 유전자의 발현 최적화 및 생물학적 활성

Optimization of the Expression of the Ferritin Protein Gene in Pleurotus eryngii and Its Biological Activity

한국균학회지 2019년 47권 4호 p.359 ~ 371
우연정 ( Woo Yean-Jeong ) - 대구대학교 대학원 자연자원학과

오시윤 ( Oh Si-Yoon ) - 대구대학교 대학원 자연자원학과
최장원 ( Choi Jang-Won ) - 대구대학교 대학원 자연자원학과

Abstract


To optimize the expression and secretion of ferritin protein associated with ion storage in the mushroom, Pleurotus eryngii, a recombinant secretion vector, harboring the ferritin gene, was constructed using a pPEVPR1b vector under the control of the CaMV 35S promoter and signal sequence of pathogen related protein (PR1b). The ferritin gene was isolated from the T-Fer vector following digestion with EcoRI and HindIII. The gene was then introduced into the pPEVPR1b secretion vector, and it was then named pPEVPR1b-Fer. The recombinant vector was transferred into P. eryngii via Agrobacterium tumefaciens- mediated transformation. The transformants were selected on MCM medium supplemented with kanamycin and its expression was confirmed by SDS-PAGE and western blotting. Expression of ferritin protein was optimized by modifying the culture conditions such as incubation time and temperature in batch and 20 L airlift type fermenter. The optimal conditions for ferritin production were achieved at 25℃ and after incubating for 8 days on MCM medium. The amount of ferritin protein was 2.4 mg/g mycelia, as measured by a quantitative protein assay. However, the signal sequence of PR1b (32 amino acids) seems to be correctly processed by peptidase and ferritin protein may be targeted in the apoplast region of mycelia, and it might not be secreted in the culture medium. The iron binding activity was confirmed by Perls’ staining in a 7.5% non-denaturing gel, indicating that the multimeric ferritin (composed of 24 subunits) was formed in P. eryngii mycelia. Mycelium powder containing ferritin was tested as a feed additive in broilers. The addition of ferritin powder stimulated the growth of young broilers and improved their feed efficiency and production index.

키워드

Agrobacterium tumefaciens -mediated transformation; Ferritin; Perinereis nuntia; Pleurotus eryngii
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