Following the completion of many genome

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1 Journal of Biomolecular Techniques 13: ABRF Automated High-Throughput Purification of 6xHis-Tagged Proteins Frank Schäfer, Ulla Römer, Melanie Emmerlich, Julia Blümer, Helge Lubenow, and Kerstin Steinert QIAGEN GmbH, Max-Volmer-Strasse 4, Hilden, Germany Methods based on immobilized-metal affinity chromatography (IMAC) technology for the isolation of 6xHis-tagged proteins offer a one-step purification process that is both robust and meets the challenge of quantitatively purifying thousands of proteins with differing structures and characteristics.to perform this method in a high-throughput, automated format, protocols have been developed that can be run on lab automation workstations. Ready-to-run protocols covering a wide range of protein purification and assay applications, and which rely on the well-established 6xHis-Ni- NTA IMAC technology, are available. An Ni-NTA magnetic bead-based protocol allows micro-scale purification of up to 15 µg of 6xHis-tagged protein per well. If larger amounts of purified protein are needed, a vacuum-controlled Ni-NTA resin-based process provides a convenient medium-scale method for purification of up to 300 µg of 6xHis-tagged protein in a 96-well format.this protocol has been adapted to further increase the yield of 6xHis-tagged proteins allowing purification of up to several milligrams of highly pure protein per well.the protocol can process 96 samples, each derived from up to 25 ml culture volume (E. coli), in less than 3 h. Examples of purification and assay applications using the ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Frank Schäfer, Max-Volmer-Strasse 4, Hilden, Germany [phone: (49) , fax: (49) , f.schaefer@de.qiagen.com]. protocols mentioned above, and data on reproducibility and cross-contamination-free processing are shown. Key Words:Automation, high-throughput, immobilized-metal affinity chromatography (IMAC), Ni-NTA, His tag. Following the completion of many genome sequencing projects, the analysis of identified open reading frames (ORFs) and the corresponding proteins has become a major research focus. To determine the structure, function, and cellular interaction maps of the identified proteins in many of these post-genomic projects, cloning of high numbers of ORFs and expression and purification of recombinant proteins is performed. 1 9 Purified proteins are then used for protein structure analysis in structural genomics studies, 6 11 for direct immobilization on microarray surfaces, 1,5, for raising antibodies by animal immunization, 1 or for functional and interaction studies. 15 Many projects require the analysis of a high number of individual proteins or variants. Therefore, a method is required that allows multiparallel processing of different proteins. This can be achieved by fusing the cdna to be expressed to a proteinaceous tag that can be used to capture the expressed proteins by a single affinity purification step. Several attempts to set up a multiwell protein purification system have been reported using maltose-binding protein (MBP) or glutathione-s-transferase (GST) protein fusions; however, the results were compromised either by yield 16 or by throughput and automation potential. 17 A two-step purification approach (TAP, tandem affinity purification) has been developed to purify and analyze protein complexes in proteomics studies; 18,19 however, TAP automation is difficult and has not been addressed. Common to the tags described above is their large size, which may contribute to the solubility of the expressed protein but which often interferes with correct folding and function of the fusion protein. Affinity tags that bind to engineered streptavidin, such as Strep-tag II, are smaller but have a relatively high dissociation constant (13 µm) 20,21 and the capacity of JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER

2 F. SCHÄFER ET AL. commercially available matrices is low (approx. 0.5 mg/ml). Other small tags include antibody tags such as the myc-tag 22 and the FLAG-tag 23 which bind to immobilized antibodies allowing the preparation of highly pure protein; however, the yield per volume of matrix is low and the costs are very high. The widely used hexahistidine tag (6xHis tag) and IMAC purification technique has many advantages. 24 The 6xHis tag is the smallest tag available (6 amino acids). It is noncharged at physiological ph and therefore usually shows the least interference with protein structure and function. 9 The high affinity of the 6xHis tag for Ni-NTA allows efficient purification on commercially available, high-capacity matrices (5-10 mg protein per ml Ni- NTA resin) at a moderate cost per preparation. Finally, purification is simple and extremely robust, with the 6xHis-tag-Ni-NTA system being the only affinity purification principle that can be performed under both native and denaturing conditions. 25,26 To keep pace with recent advances in genomic and proteomic sciences, the development of multiparallel sample processing is required. 10,27 While multiple commercially systems are available for highthroughput nucleic acid preparation, the only commercially available lab automation platform for which multiparallel protein purification protocols have been set up is the QIAGEN BioRobot series. We have set up a series of protocols for the isolation and assay of 6xHis-tagged recombinant proteins based on magnetic beads and vacuum-driven processes covering a yield range between less than 1 µg and several milligrams. These protocols and the robotics employed are described below, as are data on reproducibility, capacity, and applications. We suggest a strategy for proteomics and functional and structural genomics projects using the described purification processes. INSTRUMENTATION AND METHODS Protein Expression and Purification Chemistry Protein Expression Constructs and Cell Cultivation Plasmid constructs for bacterial expression of 6xHistagged proteins (thioredoxin in pqe-30, DHFR in pqe-40, cpn-10 in pqe-60, T7-RNA polymerase, GroES, GroEL, GroES/EL, GFP, and TNF in TAGZyme pqe-2, CAT in pproex) were transformed in E.coli M15/pREP4 and plated on LB agar containing the appropriate antibiotics. Single colonies were picked to inoculate 0.5 ml precultures in 96-well blocks. E.coli cells were grown in LB medium containing ampicillin and kanamycin at 37 C and shaken at 150 rpm in multilevel shaker incubators (Infors, Basel, Switzerland). Precultures were grown overnight and used to inoculate main cultures to an OD 600nm of 0.05 to 0.1. Main culture volumes were 1 ml in 96-well blocks, 2.5 ml in 48-well blocks, 5 ml in 24-well blocks, and 25 ml in 250 ml-erlenmeyer shake flasks. Protein expression was induced by the addition of 0.5 mm IPTG when an OD 600nm of 0.6 to 1.0 was reached, followed by incubation for 4 h (shake flasks) or 16 h (multiwell blocks) unless otherwise stated. Cells were pelleted in the blocks by centrifugation and frozen at 20 C. For the high-yield Ni-NTA Superflow 96 protocol (see below), 25-mL cultures were sedimented in the wells of 24-well blocks by repeated centrifugation. Multiwell blocks were supplied by QIAGEN. To avoid leakage from well to well during incubation, blocks were covered with gas permissive AirPore tape (QIAGEN). For reproducibility studies, 5-L cultures were grown in a B5 fermenter (B. Braun Biotech, Melsungen, Germany) and pelleted in aliquots in the multiwell blocks as required. Protein Purification Automated protein purification in multiwell formats was performed as described 28,29 (or see In brief, purification on BioRobot workstations started with lysis of bacterial cells by resuspension and shaking on the robot shaker platform. For purification under native conditions, lysis buffer NPI-10 (50 mm NaH 2 PO 4, 300 mm NaCl, 10 mm imidazole, ph 8.0) containing 400 µg/ml lysozyme was used. During lysis, the affinity resin is distributed into the appropriate multiwell vessels and equilibrated with buffer NPI-10. Depending on the protocol performed, nucleases (Benzonase) were added to the lysis assays to reduce viscosity. Unless otherwise stated, 1 3 U Benzonase (Merck, Darmstadt, Germany) was added per milliliter of culture volume. After completion of lysis, lysates were either cleared by vacuum-driven filtration on the robotic workstation (Ni-NTA Superflow 96 protocols), or processed without a clearing step (magnetic bead protocols), and 6xHis-tagged proteins were bound to the respective Ni-NTA matrix. Unbound material was washed from the resin using buffer NPI-20 (NPI-10 containing 20 mm imidazole) and 6xHis-tagged protein eluted with NPI-250 (NPI-10 containing 250 mm imidazole). Protein yield was determined by Bradford Analysis (BioRad, Munich, Germany) performed in microtiter plates on a Spectramax spectral photometer (Molec- 132 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER 2002

3 AUTOMATION OF MULTIPARALLEL PROTEIN PURIFICATION T ABLE 1 Automated Protocols for 6xHis-Tagged Protein Applications Protocol Application Culture volume Yield (per well) Workstation Ni-NTA HisSorb Assay immobilization of BioRobot 3000, 8000, up to 150 ng and 9600 Ni-NTA Magnetic Purification and 1 ml (96-well block) 3 15 µg BioRobot 3000 and 8000 Agarose Beads assay Ni-NTA Superflow 96 Purification 5 ml (24-well block) µg BioRobot 3000, 8000, and BioRobot Kit 9600 High-yield Ni-NTA 96 Superflow Purification up to 25 ml up to 2 mg BioRobot 3000 and 8000 (shake flask) ular Devices, Munich, Germany). Elution fractions were further analyzed by SDS-PAGE on 65-lane gels (CBS Scientific, Del Mar, CA). Gels were stained with Coomassie. For purification under denaturing conditions, cells were lysed in buffer B-8 M urea (8 M urea, 100 mm NaH 2 PO 4, 10 mm Tris-Cl, ph 8.0) and either cleared by filtration, or used without clearance as described for native purification conditions. After binding, nonspecifically bound material was washed away using buffer B- 4M urea (buffer B, 4 M urea) and C-4 M urea (buffer B- 4 M urea, ph 6.3), and 6xHis-tagged protein was eluted in buffer E-8 M urea (buffer B-8 M urea, ph 4.5). Assay Procedures The 6xHis-tagged thioredoxin and DHFR proteins were purified under standard native conditions as described 26 and the buffer was exchanged to NPI-20. Protein concentration was determined by Bradford assay, and the required amounts of protein were bound to Ni-NTA magnetic beads in microtiter plates as described. 28 Proteins were exposed to protein-specific polyclonal primary antibody sera from rabbit (dilution 1:1000), washed, and probed with mouse anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP, Jackson Immunoreserach, West Grove, PA) at a 1:2500 dilution. Detection reactions were performed using OPD substrate (Sigma, St. Louis, USA) according to the manufacturer s recommendations. After 15 min of reaction time the OD 450 nm was measured. Robotics All protein purification and assay processes were automated on BioRobot workstations using QIAsoft software (QIAGEN). A microprocessor-controlled shaker, a labware handling device, and two pipetting systems are integrated with the BioRobot 3000 and Filtration-based medium-scale protein purification can be performed on the BioRobot 3000, 8000, and RESULTS Micro-Scale Automated Protein Purification Fully automated protein purification protocols using Ni-NTA Magnetic Agarose Beads provide micro-scale purification of up to 96 6xHis-tagged proteins in parallel, from expression cultures grown in 96-well blocks (1 ml culture volume). The procedure involves lysis of cells, transfer of samples to a microtiter plate, binding of proteins to Ni-NTA Magnetic Agarose Beads, washing, and optional elution of up to 15 µg of 6xHistagged protein per well in 50 µl buffer (Fig. 1, Table 1). Once bound to Ni-NTA Magnetic Agarose Beads, the directed immobilization of 6xHis-tagged proteins makes them well suited for subsequent use in assay procedures (see below). Ni-NTA Magnetic Agarose Beads are separated on a 96-well magnet, which consists of an array of 24 magnetic NdFeB (neodymium-iron-boron) rods that fit between the wells of commercially available 96- well microtiter plates. 28 Each magnetic rod attracts JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER

4 F. SCHÄFER ET AL. FIGURE 1 Micro-scale protein purification procedure. Up to 96 protein minipreps are automatically prepared in 120 min using the Ni-NTA Magnetic Agarose protocol performed on a BioRobot workstation. Cell lysis is performed automatically. It is possible to process lysates cleared by centrifugation after lysis or by crude lysates. In the latter case, lysates must be treated with nucleases (Benzonase) prior to the binding step to reduce viscosity and avoid clogging of the beads. Protein purification can be performed under native (lower left) or denaturing (lower right) conditions. the beads in four adjacent wells to the sides of the wells and keeps the beads in place while the buffer is exchanged. Once removed from the magnet, the magnetic beads are easily resuspended in buffer allowing thorough washing and elution. To demonstrate the reproducibility of this procedure, the 6xHis-tagged proteins thioredoxin and dihydrofolate reductase (DHFR) were purified simultaneously in 96 wells under native or denaturing conditions using Ni-NTA Magnetic Agarose Beads, and analyzed by SDS-PAGE. Figure 2 shows the reproducibility of the procedure with an average yield of 2.7 and 2.3 µg and low coefficient of variation (CV) values of 9.8 and 11.8%, respectively. A purity greater than 95% was achieved in all fractions analyzed under both native and denaturing conditions (Fig. 2). Automated multiparallel micro-scale purification has been successfully used for solubility studies and functional characterization of purified proteins. 15,30 Interaction and Immunoassays Using Ni-NTA Coated Surfaces 6xHis-tagged proteins are immobilized on Ni-NTA via their 6xHis tags in an oriented manner and in a fully active conformation, enabling a wide variety of proteinbased, functional, interaction or diagnostic assays Using Ni-NTA Magnetic Agarose Beads, the protein binding capacity used can easily be adjusted to the amount needed for the specific interaction. The strongly magnetic beads offer an efficient method for separating any complexes formed and can be used in fully automated procedures. Because of the specificity of the 6xHis-Ni-NTA interaction, 6xHis-tagged proteins 134 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER 2002

5 AUTOMATION OF MULTIPARALLEL PROTEIN PURIFICATION FIGURE 2 Reproducibility analyses of automated purification of 6xHis-tagged proteins under native and denaturing conditions. SDS-PAGE analyses of elution fractions (5 µl each) of 60 identical samples of 0.5-mL E.coli cultures expressing 6xHis-tagged thioredoxin (upper panel) or DHFR (lower panel) purified in parallel under native and denaturing conditions, respectively. Purification was performed using the automated 96-well Ni-NTA Magnetic Bead procedure on a BioRobot 8000 workstation. can be captured directly from complex samples such as cell lysates, giving the choice of elution of purified proteins or proceeding directly with an assay protocol. 15,34 Steinert et al. 34 immobilized His-tagged cpn-10 (the yeast HSP-10 co-chaperonin), on magnetic Ni-NTA beads and were able to capture interacting GroEL (the bacterial HSP-60), which had been previously shown to form a stable complex with cpn Zimmermann and Herberg 31 used Ni-NTA coated beads to immobilize the functional C-subunit of camp-dependent protein kinase A (PKA) and to identify physiological inhibitors of PKA. 36 Purification and assay protocols can be combined for flexible and convenient adaptation to specific applications. For example, protein can be purified and subsequently assayed immunologically on the bead. 34 For standard imunoassays, signal-to-noise ratios of 20 and higher are achieved when antigen is presented in an oriented manner on Ni-NTA coated surfaces (Fig. 3). Low background values between 0.05 and 0.15 absorbance units were achieved with CV values of between 2% and 8%. Surfaces that allow a specifically oriented immobilization have clear advantages over random adsorption to polystyrene with respect to signal-to-noise ratio and signal strength. 32 Medium-Scale Automated Protein Purification For larger amounts of protein we have developed vacuum-driven Ni-NTA Superflow 96 protocols, which allow purification of up to 300 µg per well of 6xHistagged protein under native or denaturing conditions (Table 1). Up to 96 samples can be processed simultaneously. E. coli expression cultures are grown in a 5-mL culture volume, pelleted, and lysed in 24-well blocks. While the cells are being lysed on the shaker, the workstation prepares a 96-well Ni-NTA chromatography plate by transferring affinity resin into the wells of a chromatography module located on the vacuum manifold. The lysates are automatically transferred from the 24-well blocks to the wells of the 96-well filtration plate and cleared by vacuum-driven filtration directly onto the resin-filled chromatography module. The computer-controlled vacuum system applies a vacuum to the chromatography module, and 6xHis-tagged proteins bind to the Ni-NTA matrix while nontagged molecules flow through. Subsequent washing steps with imidazole-containing buffers remove most of weakly bound contaminating molecules. 6xHis-tagged proteins are then eluted into 96- well collection vessels (see Fig. 4). Several measures have been taken to eliminate cross-contamination from sample to sample and from well to well. The steel probes transferring the lysates from the 24-well blocks to the filtration module are intensively washed after every transfer. To avoid frothing and leaks from single outlets of the filtration module to adjacent wells, the lysates are overlayed with a thin layer of a foam-reducing reagent. Ethanol or low concentrations of antifoam solutions can be used. In JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER

6 F. SCHÄFER ET AL. FIGURE 3 Immunoassays of 6xHis-tagged antigens immobilized on magnetic beads. 6xHis-tagged thioredoxin (top) or DHFR (bottom) were bound to Ni-NTA Magnetic Beads at the concentration indicated (0: controls, no antigen present during binding) and detection reactions were performed using primary protein-specific antibodies and a secondary antirabbit-hrp conjugate. Color development was induced by reaction with OPD substrate. All measurements were performed in four replicate wells. CV values are given for each antigen concentration. subsequent steps the chromatography plate is placed on top of a channeling block which isolates the outlets from one another to prevent any cross-contamination (Figs. 4 and 5). We found lysis of E.coli cells using lysozyme in buffer NPI-10 to be more effective than approaches using detergents such as CHAPS, N-tetradecyl-N, N- dimethyl-3-ammonio-1-propanesulfonate (SB3-14), or octyl- -D-thioglucopyranoside. 37 Yield of the purified His-tagged proteins was up to 100% higher when the enzymatic protocol was performed (data not shown). The Ni-NTA Superflow 96 protocols allow purification of up to 300 µg of highly pure 6xHis-tagged proteins and have been developed for applications such as determination of expression rates (this can be performed under both native and denaturing conditions) and to obtain enough material for functional studies or to raise antibodies by animal immunization. Automated Protein Purification with Further Increased Yield For applications demanding higher protein yields we have adapted the Ni-NTA Superflow 96 BioRobot protocols, allowing processing of larger culture volumes and the purification of milligram amounts of Histagged protein per well (Table 1). Increasing the 136 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER 2002

7 AUTOMATION OF MULTIPARALLEL PROTEIN PURIFICATION FIGURE 4 Ni-NTA Superflow 96 BioRobot procedure. Cells were grown in four 24-well blocks in 5-mL culture volumes to allow processing of a maximum of 96 samples in parallel. Beginning with lysis, the workstation performs all steps of the purification procedure. Ninety-six protein minipreps are automatically prepared in 135 min. amount of IMAC resin used in the procedure and an optimized lysis buffer formulation enables up to 25 ml (purification under native conditions) or 15 ml (purification under denaturing conditions) of cell culture medium (LB) to be processed. The procedure is basically the same as that for the standard mediumscale process (Fig. 4) except that growth of ml E.coli cultures is performed in shake flasks. The cultures are pelleted by repeated centrifugation into 24- well blocks. To evaluate reproducibility, 6xHis-tagged green fluorescent protein (GFP) from 48 identical 25- ml samples from a single bacterial culture was purified under native conditions. SDS-PAGE from 48 elution fractions showed greater than 90% purity at an average yield of 1.3 mg and CV value of 3.4% (Fig. 6). Efficiency of 6xHis-tagged protein recovery using the Ni-NTA Superflow 96 protocols is typically between 75% and 80% (data not shown). Eight different constructs for the expression of Histagged proteins were transformed into E.coli, plated, JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER

8 F. SCHÄFER ET AL. FIGURE 5 Medium-scale purification for expression-clone screening and protein characterization.a mixture of vector constructs for the expression of proteins of 12, 15, and 28 kda, and a 48/50-kDa heterodimer, was transformed into E.coli, plated on selective medium, and 96 colonies were picked at random for inoculating 5-mL cultures. Expression of His-tagged proteins was induced with IPTG overnight. Cells were pelleted and lysed and 6xHistagged proteins purified under denaturing conditions.a 5-µL aliquot of each elution fraction (450 µl) was loaded for SDS-PAGE analysis; proteins were visualized by Coomassie staining. and six individual colonies from each transformation picked at random for inoculating 25-mL cultures. 38 As in the standard medium-scale protocol, pellets were resuspended in 0.5 ml lysis buffer containing nucleases, to reduce viscosity of the lysates. Purification of 48 samples on a BioRobot 3000 resulted in yields of up to several milligrams (Table 2) with high purity (Fig. 7). Yields of some proteins varied (see lanes marked T) because individual clones had been grown. Figure 7 shows that this technology is suitable for purification of large protein complexes such as the bacterial chaperone system GroES/GroEL which has a heptameric substructure and an overall calculated molecular weight of 910 kda (see lanes E, copurification of coexpressed untagged GroEL with 6xHis-GroES). Furthermore, as the complete process was performed using buffer permitting interaction between the heat shock proteins, 34,35 overexpression of 6xHis-GroES or 6xHis-cpn-10 alone allowed the capture of endogenous E. coli GroEL protein (lanes marked S and 10). The interaction between GroEL and cpn-10 was weaker than that between GroEL and the homologous partner GroES, as only small amounts of GroEL could be captured by cpn-10. Figure 7 also demonstrates that proteins can be obtained with high purity over a range of expression levels from 170 to 4000 µg per well (corresponding to expression rates of mg/l culture volume; compare lanes marked 10 and G). The standard version of the Ni-NTA Superflow 96 protocol has been adapted to deliver higher yields T ABLE 2 Yields of 6xHis-Tagged Proteins Using the Adapted Ni-NTA Superflow 96 Protocol 6xHis tagged protein Total yield per well (µg)* Protein concentration (mg/ml) Green fluorescent protein T7 RNA polymerase GroES Chloramphenicol acetlytransferase GroEL Tumor necrosis factor GroES/GroEL Cpn *Yield obtained in two 550-µL elution fractions (average of six independent purifications). Eighty percent of the protein elutes in the first 550 µl. Protein concentration in the first 550-µL elution fraction. 138 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER 2002

9 AUTOMATION OF MULTIPARALLEL PROTEIN PURIFICATION FIGURE 6 Reproducibility analysis using the increased-yield Ni-NTA Superflow 96 protocol. 6xHis-tagged GFP was grown in a 5-L fermenter and 25-mL aliquots were pelleted in four 24-well blocks to give identical material to all of the 96 wells. Protein was purified and 5-µL aliquots of the first elution fraction were separated by SDS-PAGE and visualized by Coomassie staining. to meet the needs of projects in structural genomics and proteomics. Milligram amounts of highly pure protein are a good starting point for screening of crystallization conditions, capturing molecule generation (antibodies, aptamers), functionally coating microarray surfaces, or obtaining material to be directly spotted onto a series of microarrays. Table 1 summarizes the protocols that have been developed for partial or fully automated multiparallel protein purification and assay procedures on various laboratory workstations. The use of Ni-NTA coated microtiter plates (HisSorb plates) has not been addressed for protein purification (Table 1), but this has been done successfully. 15 Multiparallel Cultivation of E.Coli Cultivation of the cells to be processed needs to be performed in a multiparallel mode to allow economical protein expression and purification. Cultivation in multiwell blocks is preferred to cultivation in single vessels because of the greatly simplified handling. We were interested to know how the 96-, 48-, and 24-well formats compared with each other and with shake flasks in terms of growth parameters and protein yields. Representative growth curves are shown for a single clone grown in parallel in the indicated vessel (Fig. 8). Both well-to-well and block-to-block reproducibility of growth parameters is high within every FIGURE 7 Automated purification of milligram amounts of 6xHis-tagged protein.vector constructs for the expression of 6xHis-tagged proteins were transformed into E.coli and plated on selective medium, and colonies were picked for inoculating 25-mL cultures. Expression of 6xHis-tagged proteins was induced with IPTG for 16 hr. Cells were pelleted in 24-well blocks and processed on a BioRobot 3000 workstation using 200 µl Ni-NTA Superflow resin per well, and with 2 mm ATP, 20 mm KCl, and 20 mm MgCl 2 in all buffers to allow interaction between GroES (cpn-10) and GroEL. Five microliters (0.9%) of the first elution fraction was loaded for SDS-PAGE and proteins were visualized by Coomassie staining. G, green fluorescent protein (29 kda);t, T7 RNA polymerase (100 kda) ; S, E. coli GroES (12 kda); C, E. coli chloramphenicol acetyltransferase (28 kda); L, E. coli GroEL (60 kda);, human tumor necrosis factor (18 kda); E, E. coli GroES purified as a complex with co-overexpressed nontagged GroEL (12 and 60 kda); 10, S. cerevisiae cpn-10 (10 kda); M, markers. JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER

10 F. SCHÄFER ET AL. FIGURE 8 Comparison of cell growth in multiwell blocks and flasks. An overnight preculture of E.coli M15/pREP4 carrying a pqe-30 vector construct for expression of 6xHis-tagged thioredoxin was used to inoculate LB cultures of different volumes with an OD 600nm 0.05 in the indicated vessels.all vessels were shaken at 37 C and 150 rpm. Protein expression was induced by addition of 1 mm IPTG at 4 hr. OD was measured at 600 nm at the time points indicated. cultivation system (data not shown). Growth rates are very similar in 48- and 24-well blocks and in shake flasks (Fig. 8). In addition, protein expression levels are basically the same in these vessels (Table 3). In 96- well blocks, growth curves were flatter and expression rates markedly lower. This is probably because of the reduced aeration of the culture medium, which results from the smaller surface area. DISCUSSION Protein purified using one-step affinity purification processes, such as the Ni-NTA IMAC-based processes described here, is typically at least 80 90% pure. This level of purity is sufficient for most applications, such as immunization or functional analyses, but may sometimes be insufficient for direct use in crystallization studies. 27 For crystallization, a second purification step using standard chromatography (ion-exchange, gel filtration) is often performed to achieve higher purity. Introduction of a different second tag allowing a different purification procedure directly following the initial capture step is possible, and is currently used by many researchers. 39 However, using a double-tag approach, the fusion part of an investigated protein becomes relatively long possibly affecting function and/or structure and may also require subsequent proteolytic removal of the tag portion. Using a second tag can be advantageous and form an integral part of the purification strategy, as has been shown for the tandem affinity approach. 18,19 Robotic systems for nucleic acid purification in multiwell formats are relatively commonplace; 40 however, with the exception of some lab-specific developments, the only commercially available lab automation platform for which multiparallel protein purification protocols have been set up is the QIA- GEN BioRobot series. 27 The highly flexible and robust IMAC principle upon which the protocols are based allows purification under denaturing conditions, which enables the determination of expression rates and isolation of poorly soluble molecules, such as membrane proteins 26 (see also references cited in ref. 26). Currently available protocols have been developed for processing E.coli cultures; however, some preliminary experiments have shown that they can be adapted for processing eukaryotic cells (e.g., yeast and cultured mammalian cells). The principles and procedures that we have set up for the multiparallel purification of recombinant proteins which are reviewed here have been successfully used by many laboratories in several companies and nonprofit institutions. 15,30,41 Structural genomics, which requires the expression and characterization of large numbers of proteins, is expected to provide a link between DNA sequence information and protein function Below, we suggest a high-throughput strategy for the various steps in proteomics and functional and structural genomics pro- 140 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER 2002

11 AUTOMATION OF MULTIPARALLEL PROTEIN PURIFICATION T ABLE 3 Protein Expression Eates in E.coli Cultured in Different Multiwell Blocks and in Shake Flasks Expression rate (mg 6xHis-protein per liter culture volume) Protein 96-Well block 48-Well block 24-Well block Shake flask 6xHis-CAT xHis- Thioredoxin xHis GroES/GroEL nd xHis GFP nd xHis DHFR nd, not determined. jects. Such steps include protein solubility studies, analysis of protein expression rates, and production of protein in milligram amounts using some or all of the protocols developed for automated protein purification. As a starting point, the fast, fully automated Ni- NTA magnetic bead protocols can be performed in parallel under native and denaturing conditions for expression-clone screening and solubility studies. The Ni-NTA Superflow 96 standard protocols process 5- ml E.coli cultures grown in 24-well blocks, and are suitable to quantify protein expression and enable estimation of expression rates in larger culture volumes. Furthermore, the procedure delivers µg of protein, which is sufficient for most functional studies. If more is required, the adapted Ni-NTA Superflow 96 standard protocols deliver milligram yields of protein, which enables screening of crystallization conditions, immunization of animals, or selection of aptamers to generate capture molecules for coating of microarray surfaces. 1,12,42 Alternatively, protein can be directly spotted onto microarray surfaces, with milligram amounts of purified protein generally being sufficient to generate a large number of uniform chips, allowing highly reproducible analyses. ACKNOWLEDGMENTS Sally Bee for preparing the manuscript, Sabine Rospert (MPI Halle, Germany) for providing vector for expression of Histagged cpn-10, and Jutta Drees for helpful discussions. This work was supported in part by grant D from the Bundesministerium für Bildung und Forschung (BMBF), Bonn, Germany. REFERENCES 1. Cahill D. Protein and antibody arrays and their medical applications. J Immunol Methods 2001;250: Knaust RKC, Nordlund P. Screening for soluble expression of recombinant proteins in a 96 well format. Anal Biochem 2001;297: Pizza M, Scarlato V, Masignani V, et al. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 2000;287: Larsson M, Gräslund S, Yuan L, et al. High-throughput protein expression of cdna products as a tool in functional genomics. J Biotechnol 2000;80: Walter G, Büssow K, Cahill D, Lueking A, Lehrach H. Protein arrays for gene expression and molecular interaction screening. Curr Opin Microbiol 2000;3; The Protein Structure Factory ( 7. Burley S. An overview of structural genomics. Nat Struct Biol (Suppl) 2000;7: Christendat D, Yee A, Dharamsi A., et al. Structural proteomics of an archaeon. Nat Struct Biol 2000;7: , 9. Lewis, HA, Furlong EB, Laubert B, et al. A structural genomics approach to the study of quorum sensing: Crystal structures of three LuxS orthologs. Structure 2001;9: Braun P, Hu Y, Shen B, et al. Proteome-scale purification of human proteins from bacteria. Proc Natl Acad Sci USA 2002;99: Yee A, Chang X., Pineda-Lucena, A, et al. An NMR approach to structural proteomics. Proc Natl Acad Sci USA 2002;99: Joos TA, SchrenkM, Höpfl P, et al. A microarray enzyme-based immunosorbent assay for autoimmune diagnostics. Electrophoresis 2000;21(13): MacBeth G, Schreiber SL. Printing proteins as microarrays for high-throughput function determination. Science 2000;289: Zhu, H., Bilgin, M., Bangham, R., et al. Global analysis of protein activities using proteome chips. Science 2001;293: Lanio T, Jeltsch A, Pingoud A. Automated purification of His6 tagged proteins allows exhaustive screening of libraries generated by random mutagenesis. Biotechniques 2000;29: Felleisen R, Zimmermann V, Gottstein B, Müller N. Use of a 96-well format for the affinity purification of maltose-binding protein (MBP) fusion proteins. Biotechniques 1996;20: JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 13, ISSUE 3, SEPTEMBER

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