Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, Queensland, Australia.
Protein Expression Facility, The University of Queensland, Brisbane, Queensland, Australia.
Nat Protoc. 2021 Mar;16(3):1740-1760. doi: 10.1038/s41596-020-00483-0. Epub 2021 Feb 17.
Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography. After AEP-mediated cyclization, cyclic peptides are purified by reverse-phase high-performance liquid chromatography and characterized by mass spectrometry, peptide mass fingerprinting, NMR spectroscopy, and activity assays. We demonstrate the broad applicability of this protocol by generating cyclic peptides from three distinct classes that are either naturally occurring or synthetically backbone cyclized, and range in size from 14 amino acids with one disulfide bond, to 34 amino acids with a cystine knot comprising three disulfide bonds. The protocol requires 14 d to identify and optimize a high-expressing Pichia clone in small-scale cultures (24 well plates or 50 mL tubes), after which large-scale production in a bioreactor and peptide purification can be completed in 10 d. We use the cyclotide Momordica cochinchinensis trypsin inhibitor II as an example. We also include a protocol for recombinant AEP production in Escherichia coli as AEPs are emerging tools for orthogonal peptide and protein ligation. We focus on two AEPs that preferentially cyclize different peptide precursors, namely an engineered AEP with improved catalytic efficiency [C247A]OaAEP1 and the plant-derived MCoAEP2. Rudimentary proficiency and equipment in molecular biology, protein biochemistry and analytical chemistry are needed.
环二硫键丰富的肽在药物开发和生物技术中引起了极大的兴趣。在这里，我们描述了一种在巴斯德毕赤酵母中生产环肽前体的方案，该方案使用重组天冬酰胺内肽酶（AEPs）在体外酶促成熟为环肽。肽前体带有 C 端组氨酸标签表达并分泌到培养基中，通过固定化金属亲和层析可方便地进行纯化。在 AEP 介导的环化后，通过反相高效液相色谱法纯化环肽，并通过质谱、肽质量指纹图谱、NMR 光谱和活性测定进行表征。我们通过生成来自三种不同类别的环肽来证明该方案的广泛适用性，这些环肽要么是天然存在的，要么是通过合成的骨架环化的，大小从含有一个二硫键的 14 个氨基酸到含有三个二硫键的胱氨酸结的 34 个氨基酸不等。该方案需要 14 天时间在小规模培养（24 孔板或 50 mL 管）中鉴定和优化高表达毕赤酵母克隆，然后在生物反应器中进行大规模生产和肽纯化可在 10 天内完成。我们以 Momordica cochinchinensis 胰蛋白酶抑制剂 II 为例。我们还包括在大肠杆菌中生产重组 AEP 的方案，因为 AEPs 是用于正交肽和蛋白质连接的新兴工具。我们重点介绍两种优先环化不同肽前体的 AEP，即具有改进的催化效率的工程化 AEP [C247A]OaAEP1 和源自植物的 MCoAEP2。需要基本的分子生物学、蛋白质生物化学和分析化学专业知识和设备。
