Competent Cell Preparation and Transformation of Pichia pastoris.

During the past three decades, the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii) has gained widespread acceptance as a system of choice for heterologous protein expression. One of the reasons that this yeast is used so frequently is the simplicity of techniques required for its molecular genetic manipulation. There are several different protocols available for introducing DNA into P. pastoris using electroporation or heat shock. We describe here a shortened protocol for cell preparation and transformation that works reliably with either prototrophic markers or antibiotic selection in this host. This procedure utilizes the most efficient portions of the electroporation and heat-shock transformation protocols to yield a method that is both time-saving and effective.