背景:巴斯德毕赤酵母(syn.Komagataellaphaffii)是用于生产异源糖蛋白的最高度利用的真核表达系统之一,能够进行N-和O-甘露糖基化。在这项研究中,我们展示了来自结核分枝杆菌(Mtb)的38kDa糖脂蛋白PstS-1的O-甘露糖基化重组形式在巴斯德毕赤酵母中的表达,在一级结构上与天然分泌蛋白相似。
结果:产生了没有天然脂化信号的重组PstS-1(rPstS-1)。糖蛋白表达在甲醇诱导型启动子pAOX1的控制下,分泌由α-交配因子分泌信号指导。rPstS-1的生产在挡板摇瓶(BSF)和受控生物反应器中进行。在BSF和生物反应器中都实现了高达〜46mg/L的重组蛋白的生产。从上清液中回收重组蛋白,分三个步骤纯化,实现具有98%电泳纯度的制剂。对重组蛋白的一级和二级结构进行了表征,以及它的O-甘露糖基化模式。此外,使用来自活动性结核病患者的血清抗体进行的交叉反应性分析证明了对重组糖蛋白的识别,间接表明重组PstS-1和来自Mtb的天然蛋白之间的相似性。
结论:rPstS-1(98.9%序列同一性,O-甘露糖基化,并且没有标签)是由巴斯德毕赤酵母生产和分泌的,证明这种酵母是一种有用的细胞工厂,也可用于生产其他糖基化的Mtb抗原。rPstS-1可以用作研究该分子在Mtb感染过程中的作用的工具,并开发和改进基于重组蛋白的疫苗或试剂盒,用于血清诊断。
BACKGROUND: Pichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this
study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein.
RESULTS: The recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb.
CONCLUSIONS: rPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.