1. 生物技术药物安徽省工程技术研究中心于2012.03.28日在Protein Expression and Purification杂志上在线发表论文：Purification and characterization of human IL-10/Fc fusion protein expressed in Pichia pastoris
Interleukin (IL)-10 is 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-10 and an IgG Fc fragment (hIL-10/Fc), and expressed it in Pichia pastoris. This hIL-10/Fc fusion protein was purified from the culture supernatant using MabSelect affinity chromatography and size-exclusion chromatography. The hIL-10/Fc yield was about 5 mg/L in shake flasks, with purity exceeding 95%. In addition, the hIL-10/Fc fusion protein suppressed the phytohemagglutinin -induced IFN-γ production in human peripheral blood mononuclear cells. Pharmacokinetic study also revealed that hIL-10/Fc has a prolonged circulating half-life of about 30 hours in rats. More importantly, the hIL-10/Fc fusion protein displayed highly specific biological activity, which was slightly higher than that of the commercial recombinant human IL-10 (rhIL-10). Therefore, P. pastoris is useful in the large-scale production of hIL-10/Fc fusion protein for both research and therapeutic applications.
2. 生物技术药物安徽省工程技术研究中心于2012.12.07日在Applied Microbiology and Miotechnology 杂志上在线发表论文：Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris
Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weights of 9 and 15 kDa; the 9 kDa form is a result of proteolytic maturation of the 15 kDa precursor. Recombinant 9 kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional published methods.
In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9 and 15 kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/L in fermentation, and over 95% purity was achieved with common his-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in Pichia pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.