| The methylotrophic Pichia pastoris expression systems have been successfully used for over20years for the production of heterologous proteins. Over1,000proteins have been cloned and expressed using this system. P. pastoris is easier to genetically manipulate and culture in defined medium than mammalian cells and can be grown to high cell densities. When compared to prokaryote expression system, P. pastoris could produce soluble, correctly folded recombinant proteins which have undergone all the post translational modifications, such as the formation of disulfide bond. So, lots of the proteins that cannot be expressed in E. coli have successfully been produced in P. pastoris. Additionally, because the P. pastoris has the benefits of stable genetic modification, low cost for culture and high expression level, it is becoming increasingly important in biopharmaceutical industry. In this study, several potential therapeutic recombinant proteins have been successfully expressed in P. pastoris:1. Purification and characterization of human IL-10/Fc fusion protein expressed in P. pastorisInterleukin (IL)-10is an anti-inflammatory cytokine that could be potentially applied for clinical therapy. However, its short circulating half-life in the serum limits its clinical applications. In this study, we designed a fusion protein containing human IL-10and an IgG Fc fragment (IL-10/Fc), and expressed it in P. pastoris. The IL-10/Fc yield was about5mg/L in shake flasks, with purity exceeding95%. In addition, the IL-10/Fc fusion protein displayed highly specific biological activity, which was slightly higher than that of the commercial recombinant human IL-10(rhIL-10). Pharmacokinetic study also revealed that IL-10/Fc has a prolonged circulating half-life of about30hours in rats. Therefore, P. pastoris is useful in the large-scale production of IL-10/Fc fusion protein for both research and therapeutic applications.2. Transdermal delivery of IL-10/Fc fusion protein using covalently linked TD-1peptideTD-1is an11-mer peptide that facilitates the transdermal protein delivery through intact rat skin via hair follicles. Though there are significant clinical benefits following rhIL-10administration in patients with psoriasis, systemic administration has severe side effects and limits the clinical improvement. At the basis of part1, we designed TD-1/IL-10/Fc fusion protein to treat psoriasis by local transdermal drug delivery. In this study, the purity of TD-1/IL-10/Fc expressed in P. pastoris reached95%, and the immunosuppressive activity of the TD-1/IL-10/Fc was similar to that of IL-10/Fc.Trandermal activity assay showed that TD-1/IL-10/Fc had much more potential activity to penetrate intact skin both in vivo and in vitro. These results promote us to examine the activity of TD-1/IL-10/Fc in suppressing excessive immune response and treating psoriasis in mouse models in the future.3. Production of recombinant granulysin with simple fed-batch fermentation in P. pastorisGranulysin is antiviral and broadly tumoricidal and antimicrobial. However, it is difficult to be produced in various expression systems. In this study, we first report that expression of both9kd and15kd granulysin using P. pastoris in basal salt medium at high cell density. The yield of granulysin was at least100mg/L in fermentation, with purity exceeding95%. Functional analysis showed granulysin had cytotoxic effect in a dose-dependent manner. Here, we also described a simple but robust fed-batch fermentation process for the production of granulysin which might be applied in clinical studies in the future.In summary, the methylotrophic P. pastoris is very useful to produce heterologous proteins.It has lots of significant advantages when compared to traditional sources. This study established a methylotrophic P. pastoris expression system which consisted of screening of high expression colonies, optimization of fermentation strategies and downstream process development. |
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