Spiegel Holger, Schinkel Helga, Kastilan Robin, Dahm Pia, Boes Alexander, Scheuermayer Matthias, Chudobová Ivana, Maskus Dominika, Fendel Rolf, Schillberg Stefan, Reimann Andreas, Fischer Rainer
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, Aachen, 52074, Germany.
Biotechnol Bioeng. 2015 Apr;112(4):659-67. doi: 10.1002/bit.25481. Epub 2014 Nov 24.
We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules.
我们通过鉴定并随后去除两个蛋白酶切割位点，成功优化了一种重组多亚基疟疾疫苗候选蛋白，以便在甲基营养酵母毕赤酵母中生产。在观察到补料分批发酵的培养上清液中存在蛋白质降解后，通过质谱分析了分泌的重组蛋白的主要蛋白水解片段。质谱数据表明，与酵母KEX2蛋白酶共有基序EKRE匹配的氨基酸序列发生了切割。通过在修饰变体中删除EKRE基序，该区域的切割被完全消除。生产、纯化了这种修饰变体，并用于免疫兔子，诱导出高抗原特异性抗体滴度（2×10⁶）。兔免疫血清中的总IgG在免疫荧光试验中识别恶性疟原虫寄生虫的不同阶段，表明候选疫苗具有天然折叠结构。然而，修饰变体仍然会降解，尽管降解成了不同的片段。通过质谱和N端测序进一步分析发现，在基序PEVK下游存在第二个切割位点。因此，我们去除了包括PEVK基序在内的一段17个氨基酸的序列，随后生产出全长重组疫苗候选蛋白，且无明显降解，每升培养体积的产量为53毫克。我们清楚地证明，通过识别和去除此类切割位点，可以防止毕赤酵母内源性蛋白酶对重组蛋白的蛋白水解降解。该策略对于重组亚基疫苗的生产尤为重要，因为在重组亚基疫苗生产中，产品产量和稳定性比生产大多数治疗分子所需的严格定义的天然序列更为重要。
