Kjeldsen T, Brandt J, Andersen A S, Egel-Mitani M, Hach M, Pettersson A F, Vad K
Molecular Biology and Protein Chemistry, Novo Nordisk A/S, Bagsvaerd, Denmark.
Gene. 1996 Apr 17;170(1):107-12. doi: 10.1016/0378-1119(95)00822-5.
An alpha-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).
在酿酒酵母中产生了一种α因子前导肽/胰岛素前体融合蛋白，并进行了代谢标记，以分析成熟和分泌效率。发现分泌物质的很大一部分以高糖基化未加工形式存在，表明Kex2p内肽酶成熟不完全。在双碱性Kex2p位点后引入间隔肽（EAEAEAK），形成胰岛素前体的N端延伸，大大提高了Kex2p催化效率和胰岛素前体的发酵产量。N端延伸具有一个赖氨酸，以便随后通过胰蛋白酶或溶杆菌属赖氨酸特异性蛋白酶进行蛋白水解去除。通过在延伸部分的N端添加谷氨酸来抑制从延伸部分去除谷氨酸-丙氨酸二肽的二肽基氨基肽酶A（DPAPA）活性。出乎意料的是，这种修饰的N端延伸（EEAEAEAK）在发酵过程中在赖氨酸后被部分切割。这种单碱性蛋白水解活性被证明与Yap3p有关。通过在赖氨酸之前插入脯氨酸（EEAEAEAPK）可以防止Yap3p切割。
