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Michael Bjerke, Laurie Orr, Cristopher Cowan, and Becky Godat, Promega Corporation Abstract Automated high-throughput protein purification using Promega's MagneHis Protein Purification System provides a rapid, reliable and walkaway system for the purification of bacterially expressed, polyhistidine-tagged proteins. Automated methods have been developed to purify proteins from 1mL of bacterial cell culture on Beckman Coulter's Biomek® Laboratory Automation Workstations. The MagneHis protein chemistry is easily scalable to accommodate a variety of sample volumes. Introduction Rapid exploration of novel proteins requires high-quality, easy-to-use high-throughput protein purification. Expression and purification of recombinant proteins using a polyhistidine tag is a commonly used strategy for obtaining specific highly pure proteins in large quantities. We have developed a magnetic, mobile-solid phase system to purify polyhistidine-tagged proteins from bacterial cultures in an automated walkaway manner. By automating the purification process in settings where the workflow and sample throughput warrant, scientists can shift their efforts from sample processing to data analysis. The MagneHis Protein Purification System contains a unique Cell Lysis Reagent that is complementary to automation. This component allows efficient resuspension and lysis of bacterial cell pellets, without sonication and centrifugation (1), unlike other polyhistidine-tagged protein purification systems. We present data generated from Biomek Laboratory Automation Workstations in standard 96-well plate formats. Automated Workstations Automated methods were developed on Beckman Coulter's Biomek 2000 Laboratory Automation Workstation (Figure 1) and Biomek FX Laboratory Automation Workstation (Figure 2). These liquid handlers are capable of processing 96-sample formats, moving plates and manipulating liquid reagents throughout the procedure.
Automated Procedure Polyhistidine-tagged proteins 18 to 123kDa in size were expressed in either JM109 or BL21(DE3)pLysS E. coli bacterial strains and purified using the MagneHis Protein Purification System. These proteins included RNase HI, humanized Renilla luciferase, Rnasin* Ribonuclease Inhibitor(d), thermostable firefly luciferase, methionyl tRNA synthetase and ß-galactosidase. Bacterial cultures were grown in 96-well deep-well square culture plates at 37°C (or at 25°C for cells expressing RNasin Ribonuclease Inhibitor) in 1mL of LB medium per well to < 2.0 OD600. Cells were pelleted by centrifugation. Each automated workstation was optimized to perform the following steps of the purification process. Cell Pellet Resuspension/Lysis. The 96-well, deep-well square culture plate containing the pelleted cells received 200µl of MagneHis Cell Lysis Reagent per well. The cell pellets were resuspended by a combination of tip mixing and orbital shaking for 5 minutes. Efficient resuspension of the cells required both tip mixing and shaking. Protein Binding to MagneHis Ni-Particles. For each 1mL sample of bacterial culture, 30µL of MagneHis Ni-Particles was added to an empty well of a Greiner U-bottom multiwell plate. The resuspended cell lysates were then added to the MagneHis Ni-Particles. The plates were thoroughly mixed on an orbital shaker, and the MagneHis Ni-Particles, which selectively bind the polyhistidine-tagged proteins, were captured on the MagnaBot* 96 Magnetic Separation Device (Promega Cat. No. V8151). All unbound proteins were collected as flowthrough for analysis (optional). Note that with this volume and plate type it was necessary to process half of the lysate volume at a time due to the space limitations of the wells. Washes. Three 100µL washes with the MagneHis Binding/Wash Buffer removed contaminants and residual unbound materials. During each wash, the samples were placed on the orbital shaker for efficient resuspension of the MagneHis Ni-Particles. Elution. For each sample 100µL of MagneHis Elution Buffer was added, and samples were resuspended on the orbital shaker. After shaking, the samples were separated magnetically and the supernatants saved in a fresh 96-well plate for analysis. Results Following each automated run, 20µL of the purified, eluted proteins from various wells across the plate were analyzed by SDS-PAGE. Figures 4 and 5 show purification of polyhistidine-tagged thermostable firefly luciferase using the Biomek FX (Figure 3, Panel A) or Biomek 2000 (Figure 3, Panel B). Each instrument generated purified protein with a consistent yield across the 96-well plate. Additional proteins, ranging in size from 18 to 123kDa, were also purified using the MagneHis Protein Purification System with similar results (data not shown).
Instrument Configuration and Throughput The Biomek FX workstation processed all 96 samples simultaneously due to the 96-channel POD configuration. This increased sample throughput considerably. The Biomek 2000 can pipette 8 samples at a time, thereby increasing the flexibility in the number of samples processed during a single run but decreasing overall sample throughput (Table 1). Robotic throughput varies depending on the instrument and its configuration. While it is possible to process samples faster manually than with the Biomek 2000 manual processing of 96 samples at a time is labor intensive, tedious and prone to error.
Conclusions We have automated the MagneHis Protein Purification System for the rapid purification of polyhistidine-tagged proteins on several automated workstations. The innovative MagneHis Cell Lysis Reagent eliminates the need for manual intervention (no sonication or centrifugation steps during the purification process) and allows for a truly walkaway protein purification system. Methods have been developed for the Biomek 2000 and Biomek FX automated platforms. These methods allow a quick startup in your laboratory and offer several key advantages for end-users. These advantages include a simple process that is easily automated on a variety of platforms for high-throughput applications, chemistry that is easily scaled up for larger sample volumes and high-quality purified proteins with few background bands. * All trademarks are the property of their respective owners. For Research Use Only; not for use in diagnostic procedures.
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