HIGH-LEVEL EXPRESSION AND EFFICIENT ASSEMBLY OF HEPATITIS-B SURFACE-ANTIGEN IN THE METHYLOTROPHIC YEAST, PICHIA-PASTORIS
HIGH-LEVEL EXPRESSION AND EFFICIENT ASSEMBLY OF HEPATITIS-B SURFACE-ANTIGEN IN THE METHYLOTROPHIC YEAST, PICHIA-PASTORIS
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DOI：
10.1038/nbt0587-479
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发表时间：
1987-05-01
期刊：
影响因子：
--
通讯作者：
THILL, GP
中科院分区：
文献类型：
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作者：
CREGG, JM;TSCHOPP, JF;THILL, GP
甲醇营养工业酵母Pi-chia Pastoris是大规模生产外源蛋白的宿主系统。作为该系统的一个例子，描述了乙肝表面抗原(HBs)的合成。乙肝表面抗原基因的表达受甲醇调节的启动子控制，该启动子来源于初级乙醇氧化酶基因AOX1，OFP。牧羊人。在一种新的突变株OFP中观察到最高水平的乙肝表面抗原。AOX1结构基因缺失的巴斯德酵母，代之以由AOX1启动子-乙肝表面抗原基因表达盒组成的结构。在甲醇上生长时，该菌株的可溶性蛋白质中有2-3%是乙肝表面抗原。对重组乙肝表面抗原的鉴定结果表明，几乎所有的重组乙肝表面抗原都能稳定表达。巴斯德合成的乙肝表面抗原以类似于人血清来源的乙肝表面抗原22 nm颗粒的形式存在。还介绍了从摇瓶培养到高密度发酵生产乙肝表面抗原的放大过程。一种单一的文化。在体积为240升、密度为60克/升的细胞干重中产生0.4克/升的细胞，或总计90克的乙肝表面抗原22纳米颗粒，这一数量足以接种约900万剂疫苗。
The methylotrophic industrial yeast,Pi–chia pastoris, has been developed as a host system for the large–scale production of heterologous proteins. As an example of the systems, the synthesis of hepatitis B surface antigen (HBsAg) is described. Expression of the HBsAg gene is controlled by a methanol–regulated promoter derived from the primary alcohol oxidase gene,AOX1, ofP. pastoris. The highest level of HBsAg is observed in a novel mutant strain ofP. pastorisin which theAOX1structural gene is deleted and replaced by a construction composed of theAOX1promoter–HBsAg gene expression cassette. When grown on methanol, 2–3% of the soluble protein in this strain is HBsAg. Characterization of the recombinant HBsAg indicates that virtually all of theP. pastoris–synthesized HBsAg exists in a form which is similar to human serum–derived HBsAg 22 nm particles. Scale–up of the HBsAg production process from shake–flask to high cell–density fermentor cultures is described as well. A single culture of theP. pastorisHBsAg expression strain in a volume of 240 liters and at a density of 60 grams/liter dry weight of cells produced 0.4 grams/liter, or a total of 90 grams of HBsAg 22 nm particles, an amount sufficient for approximately 9 million doses of vaccine.