Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter
Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter
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DOI：
10.1016/s0378-1119(96)00675-0
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发表时间：
1997-02-20
期刊：
影响因子：
3.5
通讯作者：
Cregg, JM
中科院分区：
文献类型：
--
作者：
Waterham, HR;Digan, ME;Cregg, JM
我们报道了从毕赤酵母中克隆的3-磷酸甘油醛脱氢酶基因（GAP）的序列。该基因被预测为编码一个35.4 kDa的蛋白质，具有显着的序列相似性，从其他生物体的甘油醛-3-磷酸脱氢酶。在巴斯德毕赤酵母中使用细菌β-内酰胺酶作为报告基因的启动子研究表明差距启动子（P-GAP）是组成型表达的，尽管其强度取决于用于细胞生长的碳源而变化。在葡萄糖生长的细胞中在P-GAP控制下的β-内酰胺酶的表达显著高于在甲醇生长的细胞中在常用的醇氧化酶1启动子（P-AOX 1）控制下的表达。作为使用P-GAP的一个例子，我们证明了在P-GAP的转录控制下合成的β-内酰胺酶通过添加羧基末端或氨基末端过氧化物酶体靶向信号而正确地靶向过氧化物酶体。P-GAP已成功用于从细菌、酵母、昆虫和哺乳动物来源合成异源蛋白，因此是巴斯德毕赤酵母中P-AOX 1的有吸引力的替代物。
We report the cloning and sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) from the yeast Pichia pastoris. The gene is predicted to encode a 35.4-kDa protein with significant sequence similarity to glyceraldehyde-3-phosphate dehydrogenases from other organisms. Promoter studies in P. pastoris using bacterial beta-lactamase as a reporter showed that the GAP promoter (P-GAP) is constitutively expressed, although its strength varies depending on the carbon source used for cell growth. Expression of beta-lactamase under control of P-GAP in glucose-grown cells was significantly higher than under control of the commonly employed alcohol oxidase 1 promoter (P-AOX1) in methanol-grown cells. As an example of the use of P-GAP, we showed that beta-lactamase synthesized under transcriptional control of P-GAP is correctly targeted to peroxisomes by addition of either a carboxy-terminal or an amino-terminal peroxisomal targeting signal. P-GAP has been successfully utilized for synthesis of heterologous proteins from bacterial, yeast, insect and mammalian origins, and therefore is an attractive alternative to P-AOX1 in P. pastoris.