摘要
【目的】提高蛋白酶K在毕赤酵母中的表达产量,建立分离纯化方法。【方法】首先对蛋白酶K密码子进行优化,将其导入毕赤酵母GS115中实现分泌表达。然后对甲醇浓度、发酵温度和p H等表达条件进行优化,再对硫酸铵沉淀、亲和层析等纯化工艺进行比对分析。【结果】蛋白酶K密码子优化后实现了在毕赤酵母中的高效表达。在甲醇量0.75%、温度25°C和p H 7.0条件下进行发酵罐培养,蛋白酶K表达量达到2.2 g/L。采用Ni-NTA亲和柱对发酵液进行纯化可以得到较好的纯化效果。【结论】密码子优化后的蛋白酶K在毕赤酵母中高效表达并可以利用Ni-NTA亲和柱进行有效分离纯化。
[Objective] In order to improve the production of proteinase K and setup purification method.[Methods] We first optimized the codons of proteinase K gene and transfered it into Pichia pastoris（GS115） to realize secretory expression.And culture conditions including methanol concentration,temperature and p H were investigated.The purification methods,such as ammonium sulfate precipitation,affinity chromatography,were further optimized.[Results] Through the optimization of codons of proteinase K gene and cultural conditions,we obtained high-level expression of proteinase K.The results showed optimal fermentation condition was supplement of methanol 0.75%,fermentation temperature 25 °C and p H 7.0.The yield of proteinase K was 2.2 g per liter under the optimized fermentation condition.The efficient purification way is Ni-NTA affinity chromatograph after comparing with the other methods.[Conclusion] The results showed that proteinase K can be expressed at high level expression in P.pastoris and can be efficient purified with Ni-NTA affinity chromatograph.
出处
《微生物学通报》
CAS
CSCD
北大核心
2014年第11期2198-2207,共10页
Microbiology China
基金
国家自然科学基金项目(No.31201879)
关键词
蛋白酶K
密码子优化
表达条件优化
分离纯化
Proteniase K
Codon optimization
Fermentation condition optimization
Purification
