Biochemical Engineering Journal 77 (2013) 177–182 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journal jou rnal h om epage: www.elsevier.com/locate/bej Regular article Production of human secreted alkaline phosphatase in suspension and immobilization cultures of tobacco NT1 cell Kazunori Nakashima a , Naomi Shibasaki-Kitakawa a,∗ , Takuya Miyamoto a , Masaki Kubo a , Toshikuni Yonemoto a , Michael L. Shuler b a Department of Chemical Engineering, Tohoku University, Sendai, Japan b School of Chemical and Biomolecular Engineering, Cornell University, USA a r t i c l e i n f o Article history: Received 4 February 2013 Received in revised form 31 May 2013 Accepted 9 June 2013 Available online 15 June 2013 Keywords: Coated gel beads Immobilization culture Plant cells Recombinant protein Protein denaturation a b s t r a c t The recombinant protein production by a plant cell culture offers several advantages including prod- uct safety and ease of the purification of secreted proteins and the capability of plants to correctly fold complex proteins. However, low productivity is generally a limitation of this system, caused by the dena- turation and degradation of the secreted proteins due to proteolytic enzyme protease in the medium. In this study, we demonstrated that the transgenic tobacco NT1 cells were immobilized in Ca 2+ -alginate gel beads coated with a cell-free gel film, which significantly improved the productivity of the recom- binant human secreted alkaline phosphatase (SEAP), about 33 times larger than that in the suspension culture. The cells immobilized in the beads were protected from hydrodynamic stress during cultivation and hence did not release the endogenous protease, which caused damage to the secreted SEAP. The immobilization culture without cell leakage could be quite effective for the production of recombinant protein. © 2013 Elsevier B.V. All rights reserved. 1. Introduction A wide range of proteins, including antibodies, hormones and vaccines, has been produced in plant cell cultures [1–4]. Com- pared with traditional systems based on microbial, yeast or animal cell fermentations, the plant cell systems offer several advantages including product safety, the ease of the purification of secreted proteins (because plant cells are grown in a simple, chemically defined medium), and the capability of plants to correctly fold com- plex proteins. However, low productivity is generally a limitation of this system. In particular, the denaturation and degradation of the secreted proteins were common problems in various reports [3,5–10], mainly due to proteolytic enzymes, i.e. protease [11]. In order to prevent the loss of product proteins, the addition of protein-stabilizing agents [6–8,12], a perfusion culture to lower the extracellular protease activity [9] and the introduction of a protease inhibitor gene [13] were proposed. Plant cell immobilization is known to improve the produc- tivities of various secondary metabolites [14–17]. It has many other advantages, such as protection of the cells from any ∗ Corresponding author at: Department of Chemical Engineering, Tohoku Univer- sity, Aoba-yama 6-6-07, Aoba-ku, Sendai 980-8579, Japan. Tel.: +81 22 795 7256; fax: +81 22 795 7256. E-mail address: naomi@rpel.che.tohoku.ac.jp (N. Shibasaki-Kitakawa). surrounding stress, easy separation of the cells and products, and reuse or continuous use of the cells. Iizuka et al. [17] reported that the maximum productivity of the secondary metabolite, scopoletin, in the batch immobilization culture of tobacco cells was about 16 times larger than that in the suspension culture. There is a possi- bility that the productivities of the recombinant proteins are also enhanced by the immobilization. However, there has been little research on the effect of the immobilization on the production of recombinant proteins in plant cell cultures [18]. In this study, the production of recombinant human secreted alkaline phosphatase (SEAP) by transformed tobacco NT1 cells was studied as a model system. The cells were immobilized in Ca 2+ - alginate gel beads coated with a cell-free gel film, which was reported to be effective in preventing cell leakage without inhibi- ting cell growth [19]. The effects of the cell immobilization on the productivity and the stability of SEAP were elucidated by compar- ing those in the suspension culture. 2. Materials and methods 2.1. Gene, plasmid, and strains The SEAP gene encodes a secreted form of the human placen- tal alkaline phosphatase (PLAP) [20]. PLAP is initially anchored to the membrane via a C-terminal 26-amino-acid-long hydropho- bic domain. SEAP lacks these amino acids sequences, allowing the 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.06.004