摘要
目的克隆表达大鼠长链2-羟酸氧化酶。方法以鼠肾cDNA为模板,扩增大鼠长链2-羟酸氧化酶基因,连接到pET-28a(+)载体上构建重组质粒pET28a-rHao2,转化表达宿主Rosetta(DE3)。利用IPTG诱导重组蛋白的表达,并通过Ni柱纯化。通过监测DCIP在605 nm处吸光度值的变化来测定其对经典底物的酶动力学参数,验证活性。结果成功构建了表达质粒pET28a-rHao2(β1)和pET28a-rHao2(β2),并诱导表达了大鼠长链2-羟酸氧化酶两种同工酶β1和β2,其对2-羟基辛酸的Km分别为(25.1±1.9)和(24.6±1.2)μmol.L-1;对2-羟基异己酸的Km分别为(24.6±2.3)和(22.4±1.6)μmol.L-1。结论成功克隆表达了大鼠长链2-羟酸氧化酶两种同工酶β1和β2,获得了纯度高,活性好的重组酶,可为其体外底物及抑制剂的筛选提供模型。
OBJECTIVE To clone and express rat long-chain 2-hydroxy acid oxidase. METHODS Rat long-chain 2-hydroxy acid oxidase gene was amplified from rat kidney eDNA, and was cloned into pET-28a（ ＋ ） vector as pET28a-rHao2 recombinant plas- mid,which was then transformed into Excherichia coil strain Rosetta（ DE3 ）. The expression of recombinant protein was induced by IPTG,and was purified by Ni-NTA purification system. The kinetic parameters of classic substrates were determined by measuring the change of DCIP in As0s to identify the enzyme activities. RESULTS The pET28a-rHao2 （ β1 ） and pET28a-rHao2 （ β2 ） recombinant plasmids were successfully constructed, and the rat long-chain 2-hydroxy acid oxidase isozymes β1 and β2 were successfully induced and expressed. The Km values of 2-hydroxyoctanoic acid were（25.1 ± 1.9） and（24. 6 ± 1.2） μmol · L-1 for the rat long-chain 2-hydroxy acid oxidase isozymes β1 and β2 respectively. The Km values of（S）-（ - ）-2-hydroxyisoeaproie acid were（ 24. 6 ± 2. 3 ） and （22. 4 ± 1.6） μmol · L-1 respectively. CONCLUSION Rat long-chain 2-hydroxy acid oxidase isozymes β1 and β2 were successfully cloned and expressed with high purity and good enzyme activities. It can be used as a model to screen the substrates and inhibitors of 2-hy- droxy acid oxidase in vitro.
出处
《中国药学杂志》
CAS
CSCD
北大核心
2013年第5期331-336,共6页
Chinese Pharmaceutical Journal
基金
国家自然科学基金资助项目(81072691)
国家重点基础研究发展973计划资助项目(2011CB710802)
关键词
长链2-羟酸氧化酶
克隆
表达
long-chain 2-hydroxy acid oxidase
cloning
expression
