Method for cloning and producing the TfiI Restriction endonuclease in E. coli

摘要

A genomic DNA library of Thermus filiformis was constructed using pBR322 as a cloning vector. The methylase selection method was used to clone the TfiI methylase gene (tfiIM). A clone carrying an active TfiI methylase was identified. After sequencing the complete TfiI methylase gene and its downstream DNA sequence, a recombinase homolog was found. Because the methylase and its cognate endonuclease gene are located in proximity to each other in a particular restriction-modification system, efforts were made to clone the upstream DNA by inverse PCR. After two rounds of inverse PCR, one open reading frame (ORF1) was found upstream of the TfiI methylase gene. This ORF1, containing a Shine-Dalgarno sequence and a TATA box on the upstream side, was cloned and expressed, and TfiI endonuclease activity was detected in crude cell extracts. It is concluded that ORF1 encodes TfiI restriction endonuclease.