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_b and the plant-derived MCoAEP2. Rudimentary proficiency and equipment in molecular biology, protein biochemistry and analytical chemistry are needed.

富含环状二硫键的肽引起了药物开发和生物技术的极大兴趣。在这里，我们描述了在毕赤酵母中生产环肽前体的协议使用重组天冬酰胺内肽酶 (AEP) 在体外酶促成熟为环状肽。肽前体用 C 末端 His 标签表达并分泌到培养基中，从而可以通过固定化金属亲和层析轻松纯化。在 AEP 介导的环化后，环状肽通过反相高效液相色谱法纯化，并通过质谱、肽质量指纹、核磁共振光谱和活性测定进行表征。我们通过从三个不同类别中生成环肽来证明该协议的广泛适用性，这些类别要么是天然存在的，要么是合成主链环化的，其大小范围从 14 个具有一个二硫键的氨基酸到 34 个具有包含三个二硫键的胱氨酸结的氨基酸债券。小规模培养（24 孔板或 50 mL 管）中的毕赤酵母克隆，之后在生物反应器中大规模生产和肽纯化可在 10 d 内完成。我们以cyclotide 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 的协议，因为 AEP 是用于正交肽和蛋白质连接的新兴工具。我们专注于优先环化不同肽前体的两种 AEP，即具有更高催化效率的工程化 AEP [C247A]OaAEP1 _b和植物衍生的 MCoAEP2。需要分子生物学、蛋白质生物化学和分析化学方面的基本技能和设备。