Abstract
In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2, and SG3 leaf section (top, middle, and base). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins.
Highlights
Recombinant proteins are commonly produced in animal cells (Andersen and Krummen, 2002; Birch and Racher, 2006)
The prostatic acid phosphatase (PAP)-IgM Fc gene was detected in the top leaves of each subculture generation (SG1, SG2, and SG3) obtained from the T, M, and BA stems harvested from tobacco transgenic plants (SG1)
This study demonstrated that the production of different subculture generations of transgenic plants did not affect the expression of the recombinant PAP-IgM Fc fusion protein
Summary
Recombinant proteins are commonly produced in animal cells (Andersen and Krummen, 2002; Birch and Racher, 2006). Production in animal cells is costly, and the cultures are susceptible to human pathogen contamination. Plants have been effectively used as expression systems for large-scale production of recombinant proteins (Larrick and Thomas, 2001; Twyman et al, 2003; Fischer et al, 2004; Daniell et al, 2009; Ko et al, 2009; So et al, 2013; Lim et al, 2015; Park et al, 2015; Kim et al, 2016), and plant-based production has many advantages over other systems, including low biomass production costs and lack of human pathogen contamination (Twyman et al, 2003). Loss of the recombinant protein during plant tissue subculture is unpredictable, and sometimes, recombinant protein expression is unstable
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