PCR DIRECTED PREPARATION AND SINGLE-STEP PURIFICATION OF HIGHLY-ACTIVE HISTIDINE-TAGGED RESTRICTION-ENDONUCLEASE HGIBI (GGWCC)

The polymerase-chain-reaction technique is used to produce fusion prot eins via deletion of any intervening piece of DNA. Here a stretch of s ix histidine codons is fused to the 3'-terminus of any defined gene us ing a standard plasmid vector or a derivative thereof. The advantage o ver existing methods is that no other amino acids besides the six hist idines are added to the protein terminus and only one oligonucleotide needs to be synthesized as special primer. Genes of interest must only be cloned in the correct orientation into a universal multilinker. Us ing just one specific primer derived from the 3'-terminus of the gene and one standard primer derived from the six histidine codons the fusi on is performed by amplifying the entire vector system as described fo r inverse PCR. As an example, we report on the modification and purifi cation of the restriction endonuclease HgiBI (GGWCC). Enzymatically ac tive protein was obtained in a single step purification under nondenat urating conditions with a purity greater than 95% according to polyacr ylamide gel electrophoresis.