Protein purification often requires multiple iterations of individual column purifications, sample fractionation and visualization, and fraction pooling for the next column. Significant time is spent developing the purification strategy and using that information for subsequent protein purification cycles. A well-designed Multi-D method utilizes optimized individual column methods to create an automated and highly reproducible multistep procedure.
Here, we show the development of an automated Multi-D method on the NGC™ Chromatography System using a typical capture, intermediate, and polish purification workflow with an N-terminal small ubiquitin-like modifier-tagged C-terminal polyhistidine-tagged superfolder green fluorescent protein (SUMO-6xHis GFPsf), referred to as GFPsf hereafter. Our method development highlights the recovery and reproducibility of the automated purification process.