来自Pyrococcus furiosus的DNA聚合酶编码基因的codon优化及其在大肠杆菌中的表达
Isa Nuryana1, Fina Amreta Laksmi2, Kartika Sari Dewi3
1Research Center for Applied Microbiology, National Research and Innovation Agency, Jalan Raya Bogor Km 46, Cibinong, Bogor, 16911, Indonesia.
Journal, genetic engineering & biotechnology
|November 21, 2023
概括
我们使用子优化在大肠杆菌中开发了一种高产的复合Pyrococcus furiosus DNA聚合酶 (Pfu pol). 纯化的Pfu pol表现出高效的DNA放大,证明了其在PCR分析中的实用性.
科学领域:
- 分子生物学分子生物学
- 生物技术是生物技术.
- 酵素工程是什么? 酶工程是什么
背景情况:
- 基因聚合酶对基于PCR的DNA合成和应用至关重要.
- 高准确度的Pyrococcus furiosus DNA聚合酶 (Pfu pol) 对于强大的测试是可取的.
- 提高蛋白产量对于更广泛的研究和应用至关重要.
研究的目的:
- 开发一种具有增加蛋白质产量的重组Pfu pol.
- 为了在大肠杆菌系统中实现Pfu pol的高水平表达.
- 通过对其合成基因的编码子优化,优化Pfu pol生产.
主要方法:
- 用对大肠杆菌表达的编码子优化进行基因合成.
- 两个阶段的蛋白质净化:亲和染色学和离子交换染色学.
- 通过西方斑点分析确认纯化的Pfu pol.
主要成果:
- 成功表达和净化复合Pfu聚合物.
- 纯化Pfu pol的近似分子量为90kDa.
- 实现了 26.8 mg/L 的纯化 Pfu 聚合物的高产量.
- 在标准PCRDNA放大中证明了功能和效率.
结论:
- 成功实现了重组Pfu pol的高水平表达.
- 开发的方法促进了E. coli中复合DNA聚合酶的合成.
- 这些发现支持对重组DNA聚合酶生产的未来研究.
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