High-Efficiency Secretory Production of Peroxidase Cla Using Vesicular Transport Engineering in Transgenic Tobacco(PLANT BIOTECHNOLOGY)

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Horseradish peroxidase isozyme Cla (HRP Cla) is widely used as a reporter enzyme in a variety of detection procedures such as enzyme-linked immunosorbent assay (ELISA) and western blotting. We previously isolated the gene encoding HRP Cla and showed that HRP Cla is at first translated as a preproprotein containing propeptides at its N- and C-termini (N-terminal secretion signal peptide and C-terminal propeptide; CTPP). The signal peptide (sp) is necessary for endoplasmic reticulum (ER) translocation and the CTPP acts as a vacuolar sorting determinant. Furthermore, HRP Cla was secreted into the culture medium from cells expressing the HRP Cla gene without the CTPP region. We optimized the secretory production system of HRP Cla in tobacco plants. To determine a suitable signal peptide for high-efficient secretion of proteins, three types of sp derived from HRP Cla (ClPsp), β-D-glucan exohydrolase (GEsp) and 38 kDa peroxidase (38Psp) were compared. GE and 38P are secretory proteins highly accumulated in the culture medium of BY2 cells. The secretion efficiency was increased by 34% and 53% when GEsp and 38Psp was used, respectively. Next, we used a translational enhancer, the 5'-untranslated region of Nicotiana tabacum alcohol dehydrogenase gene (NtADH 5'-UTR). The production of HRP Cla was increased by placing NtADH 5'UTR in front of the ORF in BY2 cells. These results indicate that the localization and expression level of recombinant proteins can be controlled by the use of propeptides and 5'UTR, respectively. Finally, high-efficiency secretory production of the HRP Cla was also achieved in transgenic tobacco.
社団法人日本生物工学会の論文
2006-08-25

High-Efficiency Secretory Production of Peroxidase Cla Using Vesicular Transport Engineering in Transgenic Tobacco(PLANT BIOTECHNOLOGY)

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