A removable spacer peptide in an alpha-factor-leader insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae
A removable spacer peptide in an alpha-factor-leader insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae
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DOI：
10.1016/0378-1119(95)00822-5
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发表时间：
1996-04-17
期刊：
影响因子：
3.5
通讯作者：
Vad, K
中科院分区：
文献类型：
--
作者：
Kjeldsen, T;Brandt, J;Vad, K
在酿酒酵母中表达了α-因子前体/胰岛素前体融合蛋白，并对其进行代谢标记，以分析其成熟和分泌效率。分泌的物质中有相当一部分是以高糖化的未加工形式存在的，表明Kex2p内肽酶成熟不完整。在二碱性Kex2p位点之后引入间隔肽(EAEAEAK)，在胰岛素前体的N端延伸，大大提高了Kex2p的催化效率和胰岛素前体的发酵产量。N-末端延伸部分以赖氨酸为特征，允许随后被胰酶或赖氨酸无色杆菌赖氨酸专一性蛋白酶去除蛋白。二肽基氨基肽酶A(DPAPA)从延伸部分去除谷氨酸-丙氨酸二肽的活性通过在延伸部分N-末端添加一个Glu-Ala而被抑制，出乎意料的是，这个修饰的N-末端延伸部分(EEAEAEAK)在发酵过程中被Lys部分切割。这种单碱基的蛋白分解活性被证明与Yap3p有关。在Lys之前插入Pro(EEAEAEAPK)可以防止Yap3p切割。
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).