Pichia pastoris Expression Plasmid Assembly Using Type IIS Restriction Enzymes

A variety of cloning techniques are commonly used for the assembly of expression cassettes used for the transformation of Komagataella phaffii and, finally, the production of recombinant proteins. Restriction endonucleases are particularly useful due to their ability to recognize and cleave specific sequences in the DNA, allowing simple and reliable cloning strategies. However, the recognition sites of type II restriction enzymes leave a trace upon digestion since they cut within the palindromic recognition site. Hence, type IIS restriction enzymes that cleave outside of their recognition site became valuable tools for convenient and seamless single- and multi-gene cloning.