Overproduction, purification and crystallization of TaqI restriction endonuclease

Abstract Under phoA promoter control, Taq I endonuclease was overproduced to 5% of Escherichia coli cellular proteins. This was achieved by fusing the endonuclease gene to the first four codons of the alkaline phosphatase signal sequence. For maximal overproduction (30% of cellular proteins), a putative 14-bp hairpin within the endonuclease coding sequence was replaced with degenerate codons. In addition, Taq I methylase was required to protect host DNA. The endonuclease was purified in sufficient amounts for crystallization.