基于识别重叠序列特性的限制性内切酶的重组表达策略研究

甲基转移酶可以保护宿主基因组DNA免受限制性内切酶的消化，根据这一特性，经典的限制性内切酶制备策略是首先通过表达与其配对的甲基转移酶来保护宿主菌株，然后再共表达限制性内切酶，即“一对一”的重组表达模式。作者设计了一个新的策略：通过共表达识别重叠序列的甲基转移酶来保护宿主菌株，以制备限制性内切酶。首先将识别重叠序列的甲基转移酶转入大肠杆菌ER2566，以保护宿主基因组DNA，然后将能识别重叠序列的多个限制性内切酶分别转入该宿主菌株，以进行限制性内切酶的重组表达，即“一对多”的重组表达模式。根据这一方法，利用甲基转移酶M. EsaDix5 I、M. Alu I和M. Hha I实现了识别重叠序列(TTAA、AGCT和GCGC)的一系列限制性内切酶的重组表达，还利用甲基转移酶M. Alu I成功实现了两个新的R. Sac I同裂酶R. EcoSP4ORF25090P和R. Gma5ORF28P的重组表达。该方法简化了对抵抗限制性内切酶消化的宿主菌株的大量筛选工作，并提供了大规模制备限制性内切酶的能力。这一方法也可用于发现其他微生物中新的同工酶或同裂酶。

Recombinant Expression of Restriction Enzymes Based on Recognition of Overlapping Sequence

Methyltransferases can protect the host genomic DNA from digestion by restriction endonucleases. Based on this property, the classical strategy for preparing restriction endonuclease involves first expressing their paired methyltransferases to protect the host strain, followed by co-expression of the restriction endonuclease, i.e. a ''one-to-one'' recombinant expression pattern. In this study, we proposed a novel strategy for generating restriction endonucleases by co-expressing a methyltransferase that could recognize overlapping sequences to protect the host strain. Initially, a methyltransferase recognizing the overlapping sequences was transferred into E. coli ER2566 to protect the host genomic DNA. Subsequently, multiple restriction endonucleases capable of recognizing the overlapping sequences were individually transferred into the host strain to achieve the recombinant expression of restriction endonucleases in a ''one-to-many'' recombinant pattern. According to this approach, a series of restriction endonucleases recognizing overlapping sequences (TTAA, AGCT and GCGC) were successfully achieved using methyltransferases M. EsaDix5 I, M. Alu I and M. Hha I, respectively. Additionally, the recombinant expression of two new R. Sac I isozymes, i.e., R.EcoSP4ORF25090P and R.Gma5ORF28P, was also successfully achieved through methyltransferase M. Alu I. This approach simplifies the labor-intensive screening process for host strains resistant to restriction endonuclease digestion, while providing the capability for large-scale preparation of restriction endonucleases. Furthermore, this method can be utilized for the discovery of new isoenzymes or isoschizomers in other microorganisms.