title: High-efficiency secretory production of peroxidase C1a using vesicular transport engineering in transgenic tobacco.
abstract: Horseradish peroxidase isozyme C1a (HRP C1a) 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 C1a and showed that HRP C1a 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 C1a was secreted into the culture medium from cells expressing the HRP C1a gene without the CTPP region. We optimized the secretory production system of HRP C1a in tobacco plants. To determine a suitable signal peptide for high-efficient secretion of proteins, three types of sp derived from HRP C1a (C1Psp), beta-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 C1a 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 C1a was also achieved in transgenic tobacco.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0101, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Matsui
authors.forename: Machiko
authors.initials: M
authors.lastname: Hori
authors.forename: Nobuko
authors.initials: N
authors.lastname: Shizawa
authors.forename: Hideki
authors.initials: H
authors.lastname: Nakayama
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Shinmyo
authors.forename: Kazuya
authors.initials: K
authors.lastname: Yoshida
mesh_terms: D020121:5' Untranslated Regions; D001709:Biotechnology / Q000379:methods*; D015152:Blotting, Northern; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006735:Horseradish Peroxidase / Q000096:biosynthesis* / Q000737:chemistry*; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D020033:Protein Isoforms; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D014026:Nicotiana / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D020033:Protein Isoforms; D021382:Protein Sorting Signals; D006735:Horseradish Peroxidase