Protein purification protocols, once established, have to be highly reproducible to guarantee the success of subsequent applications such as protein crystallography (1,2). Reproducibility is a decisive issue if a protein is difficult to crystallize, but this is true also for other structural or kinetic studies such as inhibitor screening. The critical factor is not only the degree of purity itself (3), but also the capability to maintain constant quality over extended time periods and with different operators.

To achieve the highest purity, many protein purification protocols involve sequential separation over several chromatographic columns that work according to different separation principles, optimized for a given protein. Such multidimensional protocols start with complex mixtures (mostly crude extracts or homogenates) to end up with a fraction containing the highly enriched protein of interest (4). Although the addition of an affinity tag for heterologously expressed proteins facilitates the purification procedure, several different columns may still be necessary, even when using automated high-throughput systems (2, 5,6,7,8). Such multidimensional protocols are generally based on biocompatible integrated high-performance liquid chromatography (HPLC) systems, such as the Äkta Explorer (GE Healthcare, Zurich, Switzerland). However, if each column is run separately and operated manually, it is a very time-consuming procedure with the risk of significant batch-to-batch variability due to the human factor. There are commercial solutions to automated multidimensional chromatography, but these have limitations as well. For example, the 3D Kit for Äkta systems only allows for three-dimensional (3-D) purifications, and sample volumes recovered for re-injection between columns are limited. Furthermore, the standard setup only allows connecting up to 11 buffers or solutions, which is insufficient for equilibration, run, and cleaning in place (CIP) of more than three columns.

These system limitations would necessitate manual interventions, which inevitably would lead to time loss while waiting for an operator, and batch-to-batch variability affecting the quality of the final protein preparation. To circumvent these problems, we have modified the system layout to take advantage of a certain flexibility of this system. We report here a fully automated Äkta setup for 4- and higher-dimensional protein purifications with remote control via the Internet. Based on this setup, an exemplary purification protocol is presented that is completed in only 19 h as compared with ~1 week with manual operation, and yields protein of consistently high quality.

System hardware. All products are from GE Healthcare unless mentioned otherwise. An Äkta 100 Explorer Air with a preparative, green tubing kit, including a P960 sample pump, a 10-mm UV/VIS flow cell, and a fraction collector Frac 950 was placed in a chromatography refrigerator (Unichromat 2025; Uniequip, Munich, Germany). The standard setup was modified in 10 places as follows, described in order from buffer preparation to fraction collection (for a detailed connectivity diagram, see Supplementary (Figure 1)):

Figure 1.

1. An additional 8-port valve (Cat. no. IV-908, new UniNet address 8) was installed in front of input B2.0 This allowed for connecting7 additional buffers (i.e., the solutions to run CIP procedures for all columns sequentially by a method queue) to pump B.

2. A large polycarbonate canister (20-L Nalgene Clearboy; Nalgene/Thermo Fisher Scientific, Rochester, NY, USA) for distilled water was connected via a degasser (Cat. no. VE 7510, Viscotec, Stuttgart, Germany) to inputs B1 and A1.8. The latter allows for flushing of the flow path between V6 and pump A, thus separating incompatible solvents used for CIP (e.g., pure ethanol and 8 M urea). In this setup, the inputs A1.1, A2, B1 and B2 can still be used for the standard “buffer preparation” mode of the Äkta in case of manual runs. In addition, the water canister was connected to the sample valve (V5, position 8) for flushing pump P960.