his-tag

Several automated systems have become available recently for separation of proteins and nucleic acids using various magnetic particles. Magtration® technology developed by Precision System Science (Matsudo, Japan) performs magnetic bead separation inside a specially designed pipette tip: the beads are attracted to the tip wall by a magnet and liquid is separated from the beads similar as a filtration process. A Magtration® based separation process is illustrated in the picture below: sample (1) is mixed with the beads (2), and non-bound substances are washed out, and the beads are separated (3-5), then the bound molecules are eluted from the beads (6). Based on this advanced magnetic separation technology, we have developed a fully automated system for purification of polyhistidine-tagged (His-tag) proteins from E. coli culture. This purification system utilizes the Magtration® 12GC
robot and pre-filled reagent cartridges, and offers users the flexibility to chose Ni2+/Co2+ magnetic beads from different vendors. Automatic purification protocols have been developed and preprogrammed to obtain optimal performance for both protein purification and regeneration of magnetic beads by automation. Using this automated purification system we have shown that both water-soluble and insoluble His-tag proteins can be isolated with a high purity, yield and reproducibility, even at a low level of protein expression. Up to 12 samples can be processed in parallel on the Magtration® 12GC system, which is highly suitable for a small scale protein purification. It can also be used for rapid testing of experimental conditions for protein purification (e.g. type of the beads and imidazole concentration), and screening of protein expression.

Sample Preparation Bacterial cell pellets prepared from1.5 ml or 10 ml IPTG induced culture are frozen at -80 °C. To prepare the samples for water soluble purification, the cell pellets are mixed with SureLyse^TM buffer (PSS Bio Instruments) and refrozen for at least an hour. The samples are thawed, lysozyme (20 mg/ml) and DNase I (1000 unit/ml) are added and rocked at room temperature for 20 min. The samples are diluted with SureLyse^TM buffer to a final of 600 ul for 1.5-ml pellet or 1.2
ml for 10-ml pellet for the Magtration® 12GC robotic purification with Cartridge-WS. The initial sample processing steps for purification of inclusion body His-tag proteins are identical to those for water soluble His-tag proteins, except that the samples are diluted by 8 M guanidine HCl denaturation reagent containing 5 mM DTT instead of SureLyse^TM buffer, and rocked for 30 min for the robotic purification with Cartridge-IB under the denaturing condition. Mass Spectrometry 10 µl of the robotic purified protein was trypsinized at 37º overnight using protein/trypsin ratio of ~25, acidified to pH <
2 for subsequent nanospray LC-MS/MS analysis. Nanospray LC-MS/MS analyses of the tryptic peptides were carried out using the linear ion trap LTQ (Thermo Finnigan, San Jose, CA). Briefly, peptides were first loaded onto a trap cartridge (Agilent, Palo Alto, CA) at a flow rate of 2 µL/min. Trapped peptides were then eluted onto a reversed-phase PicoFrit column (New Objective, Woburn, MA) using a linear gradient of acetonitrile (0-60%) containing 0.1% formic acid. The eluted peptides from the PicoFrit column were sprayed into an LTQ mass spectrometer equipped with a nanospray ion source. The data-dependent acquisition mode was enabled, and each survey MS scan was followed by
five MS/MS scans with dynamic exclusion option on.

Magtration-His^TM Cartridges
Sealed, pre-filled reagent cartridges for
purification of both water-soluble and
insoluble His-tag proteins have been
optimized. Both Cartridge-WS and Cartridge-
IB have identical layouts, and they can be
used in parallel for the purifications in the
Magtration® 12GC robot. This feature
permits the user to rapidly screen multiple
samples in parallel to optimize expression
conditions for yield and water solubility.

To perform His-tag protein purification by automation, 1 tag protein purification by automation, 1-12 samples, user 12 samples, user-selected magnetic beads, and selected magnetic beads, and Magtration Magtration-His His ^TM pre pre-
filled reagent cartridges are loaded into the Magtration filled reagent cartridges are loaded into the Magtration® 12GC robot. A preprogrammed protocol IC card for His 12GC robot. A preprogrammed protocol IC card for His-tag protein tag protein
purification and magnetic bead regeneration has been developed w purification and magnetic bead regeneration has been developed with various options for the users to optimize the purification ith various options for the users to optimize the purification
dependent on the level of protein expression, nature of the prot dependent on the level of protein expression, nature of the proteins, sample volume, and the type of the magnetic beads etc.

Automated purification of His-tag water-soluble protein
Using Magtration-His^TM protein purification cartridges and the Magtration® 12GC robot, up to 120 ml the bacterial cell culture can be processed in a single run. For example, His-tag human galectin-1 (HG-1) was purified from 10-ml culture pellets (Fig. 1). The average yield of HG-1 protein was 137 µg ± SD 9.1 (n=6). For 12 samples run in parallel, 1.6 mg HG-1 protein can be purified by automation in 35 min. Compared to Qiagen Ni-NTA magnetic beads, HG-1 was isolated with higher purity using Promega MagneHis^TM Ni-particles (Fig. 1). The purified HG-1 was subjected to LC-MS/MS analysis. The mass spectrometric
data (not shown) indicated that the protein is highly purified with 63% coverage of the amino acid sequence and two peaks of 16,716.7 m/z and 16,893.0 m/z. Very similar results were obtained for His-tag TNFα protein (Fig. 3): two peaks of 19,481.9 m/z and 19,655.8 m/z were observed with 65% coverage of the amino acid sequence. Importantly, all sequenced peptides mapped to either the HG-1 or the TNFα amino acid sequences, respectively, indicated a high degree of purity of each eluted protein.

Automated recharge of Ni2+/Co2+ magnetic beads for reuse We have observed that most of the Ni/Co-magnetic beads from different vendors can be recharged with a negligible loss of the beads, and Figs. 1 and 2 also show that there is basically no change in the yield of the protein upon repeated cycles with the regenerated beads.

Fig. I. SDS analysis of the purified human galectin-1
from 10 ml bacterial culture pellet using Magtration-
His^TM cartridge-WS on Magtration® 12GC system. The
purification was repeated for 5 cycle of runs with
recharging of the beads for reuse. Duplicate samples
were used. Only the 1st , 3rd and 5th cycle of the runs
are shown; L, crude lysate; and F, flowthrough
fraction. The Nickel magnetic beads were recharged
after each purification by automatic protocol using the
same cartridges on the robot.

Fig. 2. SDS-PAGE analysis of His-tag Tumor Necrosis Factor
(TNFα) purified by automation using Magtration-His^TM Cartridge-
WS. (A) His-tag TNFα was purified from 1.5 ml of bacterial
culture pellet using Promega MagneHis^TM Ni2+ beads (Lane 1) or
Dynal Talon^TM Co2+ (Lane 2) magnetic beads. (B) His-tag TNFα
was purified for three cycles of the run, and the Talon^TM Co2+
magnetic beads were recharged and reused after each of the
purification run with triplicates of 1.5 ml bacterial culture samples.

Fig. 3. LC-MS/MS analyses of the tryptic peptides of TNFα
purified by automation on Magtration® robot system using
the Co2+ magnetic beads and the prefilled reagent cartridge-
WS. The data-dependent acquisition mode was enabled,
and each survey MS scan was followed by five MS/MS
scans with dynamic exclusion option on. The spray voltage
and ion transfer tube temperature were set at 1.8 kV and
160ºC, respectively. The normalized collision energy was set
at 35%.

Screening of His-tag protein at low expression levels
His-tag SPT protein can be readily purified by automation at very low protein concentrations using the pre-filled reagent
Cartridge-WS (Fig. 4). This data demonstrates that a small amount of culture sample with low level of protein expression can be purified using Cartridge-WS.

Automatic purification of His-tag protein under denaturing conditions
His-tag IL-10 polypeptides were highly purified using the Magtration-His^TM Cartridge-IB and the 12GC robot. The purity and yield of the protein upon three repeated cycles are very similar indicating that under denaturing conditions the magnetic beads can be recharged and reused for the purification.

Fig. 4. Purification of His-tagged serine palmitoyl transferase (SPT) on Magtration® 12GC using Magtration-His^TM Cartridge-WS and MagneHis Niparticles. For spiking experiments, a crude lysate from bacterial expressing SPT was mixed with a lysate of uninduced bacteria to represent 1% or
5% of the SPT crude lysate in total lysates. The fractions of L (the total lysate with spiking), F (flowthrough), and E (elution) were analyzed by
SDS-PAGE with Coomassie staining.

Fig. 5. Purification of His-tag mouse Interleukin-10 (IL- 10) from inclusion bodies under denature conditions on Magtration® 12GC system using Magtration-His^TM Cartridge-IB. The protein was purified for three cycle of
the runs, and the Ni2+ ion MagneHis^TM beads were recharged and reused for the purification. Samples are triplicates of 10 ml bacterial culture pellets, and the eluted IL-10 were analyzed by SDS-PAGE.

The Magtration® technology based bio-robot is highly suitable for protein purification with high efficiency. We have developed Magtration-His^TM pre-filled reagent cartridges for purification of both water-soluble and insoluble His-tag proteins by automation on the robotic system. The automated protocols have been developed with the optimized experimental conditions for His-tag protein purification. Multiple samples can be processed rapidly in parallel. This novel protein purification system provides the users with a powerful tool not only for small-scale protein purification but also for quick screening of protein expression.