水热
聚合酶
分子生物学
聚合酶
DNA聚合酶
热启动PCR
生物
聚合酶链反应
重组DNA
DNA
遗传学
基因
套式聚合酶链反应
作者
Fina Amreta Laksmi,Kartika Sari Dewi,Isa Nuryana,Siti Eka Yulianti,Kharisma Panji Ramadhan,Moch Irfan Hadi,Yudhi Nugraha
标识
DOI:10.1016/j.ab.2024.115581
摘要
A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the L-rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system.
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