The plant-based expression systems are now accredited as bioreactors for the high production of various biopharmaceuticals. However, low levels of agglomeration and the absence of effective procedures for purification of recombinant proteins have remained two essential obstacles in molecular farming. In this research, we have studied the production of human interferon gamma (hIFN-γ) in tobacco and analyzed the effects of elastin-like polypeptide (ELP) tag and subcellular localization on its accumulation. We report a remarkable enhancement of accumulation of the fusion proteins versus the corresponding unfused hIFN-γ proteins. Furthermore, the hIFN-γ (with and without ELP) accumulated to higher levels in the endoplasmic reticulum. The ELP fusion proteins were successfully recovered from total soluble protein with adding 2.75 M NaCl and three rounds of inverse transition cycling (ITC). The hIFN-γ was also separated from ELP with Enterokinase cleavage of the fusion protein and recovered by ITC. Inverse transition analysis indicated that the hIFN-γ-ELP variants aggregate above their inverse transition temperature and at high ionic strength. Investigation of glycosylation revealed that fused or unfused hIFN-γ proteins are N-glycosylated in different cellular locations. Moreover, N-glycosylation analysis and bioassay showed that fusion to ELP does not disturb glycosylation process and antiviral activity of hIFN-γ.

基于植物的表达系统现已获得生物反应器的认可，可用于大量生产各种生物药物。然而，低水平的团聚和缺乏用于纯化重组蛋白的有效程序仍然是分子农业中的两个主要障碍。在这项研究中，我们研究了烟草中人干扰素γ（hIFN-γ）的产生，并分析了弹性蛋白样多肽（ELP）标签和亚细胞定位对其积累的影响。我们报道融合蛋白相对于相应的未融合的hIFN-γ蛋白的积累显着增强。此外，hIFN-γ（有和没有ELP）在内质网中积累到更高的水平。加2可以从总可溶性蛋白中成功回收ELP融合蛋白。75 M NaCl和三轮反向过渡循环（ITC）。还通过肠激酶切割融合蛋白将hIFN-γ与ELP分离，并通过ITC回收。反向跃迁分析表明，hIFN-γ-ELP变体在其反向跃迁温度以上和高离子强度下聚集。糖基化研究表明，融合或未融合的hIFN-γ蛋白在不同细胞位置被N-糖基化。此外，N-糖基化分析和生物测定表明与ELP融合不会干扰hIFN-γ的糖基化过程和抗病毒活性。反向跃迁分析表明，hIFN-γ-ELP变体在其反向跃迁温度以上和高离子强度下聚集。糖基化研究表明，融合或未融合的hIFN-γ蛋白在不同细胞位置被N-糖基化。此外，N-糖基化分析和生物测定表明与ELP融合不会干扰hIFN-γ的糖基化过程和抗病毒活性。反向跃迁分析表明，hIFN-γ-ELP变体在其反向跃迁温度以上和高离子强度下聚集。糖基化研究表明，融合或未融合的hIFN-γ蛋白在不同细胞位置被N-糖基化。此外，N-糖基化分析和生物测定表明与ELP融合不会干扰hIFN-γ的糖基化过程和抗病毒活性。