전사체 분석을 통한 Streptococcus parauberis 상시 고발현 프로모터 발굴 및 재조합 단백질 발현 최적화
Transcriptome-guided identification of novel constitutive promoters in Streptococcus parauberis and their application to recombinant protein expression
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Streptococcus parauberis is a gram-positive coccus that causes pericarditis and myocarditis in fish, posing a significant threat to the aquaculture industry. However, there is a lack of studies on the development of genetic tools for creating prophylactic measures against the disease caused by S. parauberis. Therefore, we characterized the native promoter for protein expression and ultimately engineered S. parauberis as a vaccine. In this study, we analyzed the RNA-seq data of S. parauberis at different time points (1 hour and 4 hours) and found that three genes (rbsC, EbgC, and Dxbp) were highly expressed at 4 hours compared with 1 hour of culture. We then cloned the three promoters and used the reporter green fluorescent protein to screen for the most potent promoter. The rbsC promoter showed significantly greater GFP expression than the EbgC and Dxbp promoters. The rbsC promoter was further analyzed using a bacterial promoter prediction tool which are called SAPPHIRE and phiSITE which predicted the -35 and -10 elements. Mutation of the -35 element and -10 element resulted in low or absent GFP expression compared with the wild-type sequence. Finally, we cloned the olive flounder IFN-γ gene under the control of the RbsC promoter and transformed it into E. coli BL21(DE3). After incubation at 18°C and 37°C, we confirmed the protein expression using SDS-PAGE and western blot. In summary, we discovered a novel constitutive promoter derived from S. parauberis, characterized its regulatory elements, and demonstrated its utility for heterologous pro- tein expression. This work represents a foundational step toward engineering S. parauberis for vaccine development.
Abstract
Streptococcus parauberis is a gram-positive coccus that causes pericarditis and myocarditis in fish, posing a significant threat to the aquaculture industry. However, there is a lack of studies on the development of genetic tools for creating prophylactic measures against the disease caused by S. parauberis. Therefore, we characterized the native promoter for protein expression and ultimately engineered S. parauberis as a vaccine. In this study, we analyzed the RNA-seq data of S. parauberis at different time points (1 hour and 4 hours) and found that three genes (rbsC, EbgC, and Dxbp) were highly expressed at 4 hours compared with 1 hour of culture. We then cloned the three promoters and used the reporter green fluorescent protein to screen for the most potent promoter. The rbsC promoter showed significantly greater GFP expression than the EbgC and Dxbp promoters. The rbsC promoter was further analyzed using a bacterial promoter prediction tool which are called SAPPHIRE and phiSITE which predicted the -35 and -10 elements. Mutation of the -35 element and -10 element resulted in low or absent GFP expression compared with the wild-type sequence. Finally, we cloned the olive flounder IFN-γ gene under the control of the RbsC promoter and transformed it into E. coli BL21(DE3). After incubation at 18°C and 37°C, we confirmed the protein expression using SDS-PAGE and western blot. In summary, we discovered a novel constitutive promoter derived from S. parauberis, characterized its regulatory elements, and demonstrated its utility for heterologous pro- tein expression. This work represents a foundational step toward engineering S. parauberis for vaccine development.