Introduction
Affinity purification is a powerful tool to enrich proteins of interest for proteomic analysis. We have developed a fully automated Magtration® 12GC system for the multi-parallel protein purification. Up to 12 samples can be processed simultaneously on the system using magnetic beads in suspension or chromatographic beads packed into a Tip column.
Reagents for protein purification and the bead or column regeneration are pre-dispensed in a sealed
cartridges for the robotic runs. There is only a minimum reagent handling required. Protocols for protein purification have been optimized and preloaded into a protocol card to control the automation process. High protein purity and yield were obtained on this system. For small scale His-tag protein purification and the protein expression screening, Ni2+ or Co2+ magnetic beads were used on the robotic system. We have also introduced nickel chromatographic resin packed Tip-columns to scale up the protein purification yield. 66 mg of His-tag green fluorescent protein can be purified by processing 12 samples from a total of 1.5 liter bacterial culture on the Tip-columns on the robotic system in 45 mins. A high purification yield can be achieved by using the Tip-columns processed in parallel on the robot. The robotic system can also be used for purification of GST-fusion proteins using glutathione coupled magnetic beads. 1.2 mg of GST-Rho4p can be purified from about 250 ml bacterial culture. The same robotic system has been extended to IgG purification using protein A coupled magnetic beads. A total of 3.2 mg IgG can be purified in less than an hour by processing 360 ul of human serum on the robot. We here present a fully automated platform,
which can be used for multi-parallel affinity purification and expression screening of proteins.

Materials and Methods
1. Bacterial cell lysis: Bacterial cell pellets are lysed using the SureLyse^TM Reagent (PSS Bio Instruments) and the Enzymatic treatment using lysozyme and DNase I. For preparation of bacterial lysates expressing GST-Rho3p and GST-Rho4p, EDTA-free protease inhibitors (Roche) were added to the lysate.
2. Pre-filled reagent cartridges and consumables (Fig. 1A, B and C): Reagents for the protein purification and
magnetic beads regeneration are pre-filled into a cartridge sealed by aluminium foil and can be inserted into the reagent cartridge rack on the robot for the automated runs. It provides minimum pipetting work for reagent preparation. Consumables such as Magtration® pipette tips, tip-sheaths and 1.5-ml or 2-ml micro-tubes are used for the automated runs.
3. Magnetic beads: MagneHis^TM Ni-particles used for His-tag protein purification and the MagneGST^TM particles used for GST-fusion protein purification were purchased from Promega. A suitable type and amount of the magnetic beads can be selected from different vendors to achieve purification by automation with high yield. The beads used for purification can be regenerated by automation for its reuse.
4. Tip-columns (Fig. 1D): Ni Sepharose Fast Flow resins (GEHC) were packed into a PSS designed Tip to a bed volume between 0.2 – 0.5 ml. The Tip-column can be used for a large scale protein purification by automation on a robot.
5. Robotic Operation Procedures:
a) Set up the consumables, pre-filled reagent cartridges and cell lysates on a Magtration® 12GC robot.
b) Insert a protocol IC card preloaded with different purification protocols into the robot.
c) Select a “Protein Purification” protocol and follow the options from the menu screen such as sample size, elution volume, imidazole concentration (His-tag protocol only) and then START the purification protocol.
d) After the purification, select “Bead Regeneration” or “Column Regeneration” protocol to start the regeneration.

Purification & Expression Screening of His-tag proteins using Ni2+/Co2+ Magnetic beads
Using the Magtration-His^TM purification cartridges and Magtration® 12GC robot, up to 120 ml of the bacterial cell culture can be processed in a single run in 35 mins. His-tag water-soluble proteins and inclusion bodies were highly purified on the robot using Magtration-His^TM purification Cartridge-WS and Cartridge-IB, respectively. Promega MagneHis^TM Ni magnetic particles were used for the protein purification. The His-tag proteins can also be purified using Dynal Talon^TM Co2+ magnetic beads on
the robot. The Ni2+/Co2+ magnetic beads used for purification were regenerated by automation process and reused for several times for the protein purification.

Fig. 2A : His-tag protein HG-1 from 8 ml culture pellet was purified using Magtration-His^TM cartridge-WS on the robot. The purification was performed in duplicates for up to 5 cycles with the recharging of the beads for the reuse. M,Markers; Crude Lysate (Lane 1); Flow through fraction (Lane 2) ;Elution fractions in duplicates for cycle 1(Lanes 3 and 4), cycle 3 (Lanes 5 and 6) and cycle 5 (Lanes 7 and 8) of purification.
Fig. 2B : His-tag protein gag p-24 from 8 ml culture pellet was purified using Magtration-His^TM cartridge-IB on the robot. The purification was performed in duplicates for up to 3 cycles with the recharging of the beads for the reuse. Elution fractions in duplicates for cycle 1(Lanes 1 and 2), cycle 2 (Lanes 3 and 4) and cycle 3 (Lanes 5 and 6) of purification.

Scale-up Purification of His-tag proteins using Tip-column packed with nickel resins
Nickel Sepharose Fast flow resins (GEHC) packed into a PSS designed tip has been used for large-scale purification of His-tag proteins by automation. Up to 66 mg of His-tag green fluorescent protein can be purified by processing 1.5 liters of bacterial culture in a single run on the Magtration® 12GC robot. Automated protocol provides users with option for selecting different imidazole concentration. Tip-column can be regenerated by automation and re-used for several times.

Fig. 3 : Green Fluorescent protein (GFP) from 125 ml culture pellet was purified using Tip-column on the 12GC robot with 20mM and 30 mM imidazole in binding & wash buffer. Crude Lysate (Lane 1 in Figs. 3A and B); Flow through fractions (Lane 2 in Figs. 3A and B); Elution fractions 1 to 5 (Lanes 3 -7 in Figs. 3A and B). A total of 5 elutions of 600 µl is performed on the robot while using Tip-column for protein purification.

Glutathione S-transferase (GST) fusion proteins were purified using the protein purification system. 8 ml samples were processed in parallel on the Magtration® 12GC robot using Magne-GST beads (Promega). 30 µg of GST-Rho3p and 50 µg of GST-Rho4p were purified at the low level of the protein expression (Fig 4). Additional studies using lysates prepared from 20 ml GST-Rho4p culture pellets gave approx. 107 µg
protein. A protocol for the regeneration and reuse of the glutathione-magnetic beads has also been developed. The magnetic beads can thus be reused at least three times for the purification of same proteins.

IgG from Human serum as well as various species such as guinea pig serum, rabbit serum, mouse ascites fluid can be purified using protein A magnetic beads on the Magtration® 12GC robot. Approx. 270 µg of human IgG was purified from 30 µl human serum to a high purity. Total of up to 3.2 mg of human IgG can be purified by running 12 samples in parallel on the robot. Protein A magnetic beads were regenerated also by automation and reused for 5 cycles. The automated purification of IgG from guinea pig and rabbit sera gave low yield and less purity (Fig. 6). Monoclonal antibody of mouse ascites fluid was purified to 115 µg from 90 µl sample (Fig. 6, lane 9).

􀂃 We provide a fully automated protein purification system containing a Magtration® 12GC bench-top robot, pre-filled reagent cartridges, and the optimized preloaded protocols.
􀂃 Users can simply select different experimental conditions from the menu screen of the robot to start an automated run for protein purification. There is a minimum requirement for labor work and protein purification knowledge.
􀂃 Multiple protein samples can be processed in parallel using both magnetic beads in suspension and chromatographic resins in a Tip-columns on the automated system.
􀂃 His-tag proteins, GST-fusion proteins and IgG can be rapidly purified to high purity, yield and reproducibility on the robotic system. This automated system can also be extended to purify proteins by using reverse phase and ionexchange magnetic beads or chromatographic Tip-columns.

Protein	Culture volume	Protein amount1	Total protein amount2


HG-1	8 ml	178 µg	2.13 mg
gag p-24	8 ml	198 µg	2.37 mg

Protein	Culture volume	Protein amount1	Total protein amount2
GFP	80 ml	3.4 mg	40.8 mg
GFP	125 ml	5.5 mg	66 mg