Bonic Anela, Mackin Robert B
Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178-0405, USA.
Protein Expr Purif. 2003 Mar;28(1):15-24. doi: 10.1016/s1046-5928(02)00653-8.
To examine the cleavage specificity of different members of the furin/propeptide convertase (PC) family of enzymes, we have selected proglucagon (PG) as a model substrate. PG was selected because it is subject to differential processing in vivo. PG is thought to be cleaved initially at an interdomain site to produce glicentin and the major proglucagon fragment (MPGF). These intermediates are subsequently cleaved, most likely by the convertases PC2 and PC1, respectively. To determine the exact sites within PG that are cleaved by PC1 and PC2, we attempted to produce milligram quantities of human PG, glicentin, and MPGF for use in an in vitro conversion assay. A methionine residue was added to the N-terminus of each protein to initiate translation. Purification was achieved using cation exchange and reversed-phase chromatography, and the integrity of the methionylated proteins was confirmed by both electrospray ionization-mass spectrometry and amino acid analysis. The combined expression and purification scheme is fast, efficient, and results in milligram quantities of > or =95% pure proglucagon, > or =95% pure MPGF, and > or =93% pure glicentin. These prohormones are cleaved by PC1 to produce product peptides consistent with the processing of PG observed in vivo, and should therefore be suitable for further analysis of the post-translational processing of PG.
为了研究弗林蛋白酶/前肽转化酶（PC）家族不同成员的切割特异性，我们选择了胰高血糖素原（PG）作为模型底物。选择PG是因为它在体内会经历不同的加工过程。据认为，PG最初在一个结构域间位点被切割，产生肠高血糖素和主要胰高血糖素原片段（MPGF）。这些中间体随后分别最有可能被转化酶PC2和PC1切割。为了确定PG中被PC1和PC2切割的确切位点，我们试图生产毫克量的人PG、肠高血糖素和MPGF，用于体外转化试验。在每种蛋白质的N端添加了一个甲硫氨酸残基以启动翻译。使用阳离子交换和反相色谱法进行纯化，通过电喷雾电离质谱和氨基酸分析确认了甲硫氨酸化蛋白质的完整性。联合表达和纯化方案快速、高效，可产生毫克量的纯度≥95%的胰高血糖素原、纯度≥95%的MPGF和纯度≥93%的肠高血糖素。这些激素原被PC1切割，产生与体内观察到的PG加工过程一致的产物肽，因此应适用于PG翻译后加工的进一步分析。
