Automated purification of recombinant proteins: combining high-throughput with high yield.

Abstract Protein crystallography, mapping protein interactions, and other functional genomic approaches require purifying many different proteins, each of sufficient yield and homogeneity, for subsequent high-throughput applications. To fill this requirement efficiently, there is a need to develop robust, automated, high-throughput protein expression, and purification processes. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli ( E. coli ). The first is a filtration separation protocol in which proteins of interest are expressed in a large volume, 800 ml of E. coli cultures, then isolated by filtration purification using Ni–NTA–Agarose (Qiagen). The second is a smaller scale magnetic separation method in which proteins of interest are expressed in a small volume, 25 ml, of E. coli cultures then isolated using a 96-well purification system with MagneHis Ni 2+ Agarose (Promega). Both workflows provided comparable average yields of proteins, about 8 μg of purified protein per optical density unit of bacterial culture measured at 600 nm. We discuss advantages and limitations of these automated workflows, which can provide proteins with more than 90% purity and yields in the range of 100 μg to 45 mg per purification run, as well as strategies for optimizing these protocols.