Lin, C.T., Moore, P.A., Auberry, D.L., Landorf, E.V., Peppler, T., Victry, K.D., Collart, F.R. and Kery, V.
Notes: The authors compared two methods to purify a subset of 236 hypothetical hexahistidine-tagged Shewanella oneidensis proteins: a large-scale, lower-throughput filtration separation protocol using Ni-NTA Superflow columns (Qiagen) and a lower-scale but higher-throughput magnetic bead-based purification protocol using MagneHis™ Ni-Particles. They examined several factors that can affect yield and efficiency of protein binding to these two matrices, including steric factors, protein size, amount of cell lysate and cell lysis protocol. They concluded that both matrices seem to have similar protein-binding capacities, and the larger-scale and lower-scale methods resulted in 8.7µg/OD600 and 8.8µg/OD600, respectively. When examining native proteins of similar sizes, they found binding differences between monomeric and oligomeric forms, most likely due to steric hindrances around the polyhistidine tag. However, they found no correlation between protein yield and protein size (as measured in kDa or as Stokes radius). The authors calculated that the maximum yield was approximately 200µg of protein from a lysate load of 30 OD600 and observed that some proteins are difficult to elute from the MagneHis™ Ni-Particles. Inefficient elution can affect yield. About 30% of proteins were purified to >90% homogeneity and about 40% to >80% homogeneity using MagneHis™ Ni-Particles. Fourteen of the proteins were not expressed in sufficient quantity for protein purification. Finally, the authors concluded that the automated filtration process does not save much time or labor compared to the manual filtration process, and the lower-scale MagneHis™ protocol is efficient with minimal error rate. The authors used the Biomek® FX automated workstation to process 96-well plates of E. coli cultures expressing the various S. oneidensis proteins. (3848)