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基于密码子优化的蛋白酶K在毕赤酵母中的表达及分离纯化
引用本文:孙风敏,韩焱,李文利.基于密码子优化的蛋白酶K在毕赤酵母中的表达及分离纯化[J].微生物学通报,2014,41(11):2198-2207.
作者姓名:孙风敏  韩焱  李文利
作者单位:1. 大连理工大学 生命科学与技术学院 辽宁 大连 116023;2. 大连市疾病预防控制中心 辽宁 大连 116021;1. 大连理工大学 生命科学与技术学院 辽宁 大连 116023
基金项目:国家自然科学基金项目(No. 31201879)
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。

关 键 词:蛋白酶K  密码子优化  表达条件优化  分离纯化

Expression and purification of codon optimized proteinase K in Pichia pastoris
SUN Feng-Min,HAN Yan and LI Wen-Li.Expression and purification of codon optimized proteinase K in Pichia pastoris[J].Microbiology,2014,41(11):2198-2207.
Authors:SUN Feng-Min  HAN Yan and LI Wen-Li
Institution:1. School of Life Science and Biotechnology, Dalian University and Technology, Dalian, Liaoning 116023, China;2. Dalian Center for Disease Control and Prevention, Dalian, Liaoning 116021, China;1. School of Life Science and Biotechnology, Dalian University and Technology, Dalian, Liaoning 116023, China
Abstract: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 pH 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 pH 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.
Keywords:Proteniase K  Codon optimization  Fermentation condition optimization  Purification
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