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bc04-11-20.9.rz 24.09.2001 16:17 Uhr Seite 27 In-vitro Protein Production for Structure Determination with the Rapid Translation System (RTS) Ho S. Cho1*, Jeffrey G. Pelton1, Weiru Wang2, Hisao Yokota1, and David E. Wemmer1,2 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA/USA 2 Department of Chemistry, University of California, Berkeley, CA/USA *corresponding author: [email protected] 1 The goal of the Berkeley Structural Genomics Center is to determine the structures of all proteins encoded in the genomes of Mycoplasma pneumoniae and Mycoplasma genitalium or structural homologs from other organisms. To achieve this goal, the Berkeley Structural Genomics Center is developing high-throughput methods for protein expression for use in X-ray and NMR structure determination. In collaboration with Roche Molecular Biochemicals, we are investigating the usefulness of the RTS for in-vitro protein production to generate target proteins in quantities suitable for structure determination. Very encouraging results have been obtained with the test protein phosphoserine phosphatase from Methanococcus jannaschii. The structure of the phosphoserine phosphatase (PSP) from Methanococcus jannaschii has been determined previously [1]. In this study, PSP was produced in milligram quantities with the RTS, and the data collected by X-ray and NMR for structure determination of PSP were comparable with those of PSP produced in E. coli. Two labeling methods for NMR employing 15N-algal amino acids and 15N-glycine were compared. Materials and Methods Cloning of PSP-pIVEX The gene for PSP was excised from a pET-21a construct [1] and ligated into pIVEX 2.3-MCS (Roche Molecular Biochemicals) between the Nde 1 and Bam H1 restriction sites. The new construct, PSP-pIVEX, was verified by PCR and restriction fragment analysis. The PSP-pIVEX plasmid DNA was amplified in a 1 l LB culture of DH5-α E. coli cells and isolated with a plasmid maxi kit. The final concentration of the purified plasmid was approximately 0.37 mg/ml. Expression of PSP in RTS The RTS 500 E. coli HY Kit was used to express labeled protein for NMR. The RTS 500 E. coli Circular Template ROCHE MOLECULAR BIOCHEMICALS http://biochem.roche.com Kit was used to compare the yield to the RTS 500 E. coli HY Kit. The Ho S. Cho lyophilized components of the RTS 500 E. coli HY Kit were reconstituted according to the product instructions. 15 µg of PSPpIVEX plasmid DNA were used for a 1-ml reaction. The 1-ml reaction was continuously dialyzed against a 10-ml feeding mixture, containing additional energy sources and amino acids, in the RTS Instrument set to 30 °C, with a stirring speed of 120 rpm. After 20 hours of incubation, a 5-µl aliquot was removed and checked for protein production on a 15 % SDS-PAGE gel stained with Coomassie blue. The reaction was stopped at 24 hours, and the reaction mix was transferred to a 1.5-ml tube and frozen at –20 °C until purification. RTS Introduction Purification of PSP from a 1-ml RTS reaction The 1-ml RTS reaction mixture was diluted with 2 ml of 50 mM Tris at pH 7.5 and separated into three 1.5-ml tubes. The samples were incubated at 70 °C for 30 minutes to precipitate the E. coli proteins. The precipitate was pelleted by centrifugation at 13,000 rpm for 15 minutes in a microcentrifuge. The supernatant was combined and placed in a dialysis bag with a molecular weight cut-off (MWCO) of 6 - 8 kD, and dialyzed against 1 l of 20 mM Tris at pH 8.4 for 4 hours. After dialysis, the sample was passed through a 1-ml Hightrap-Q column (Pharmacia) equilibrated with 20 mM Tris, pH 8.4. The column was washed with 5 ml of 20 mM Tris, pH 8.4. The flow through and wash were collected in 1 ml fractions. 5 µl of each fraction was checked on a 15 % SDS-PAGE gel with Coomassie staining. Fractions containing PSP were combined and concentrated to 300 µl in a 4-ml Ultrafree spin concentrator (Millipore) with a 5-kD MWCO membrane. The buffer was exchanged twice by adding 3 ml of a solution containing 20 mM Tris at pH 7.5, 300 mM NaCl, 1 mM EDTA, and 10 mM DTT, and reconcentrating to 300 µl. The sample was then transferred to a 500-µl Ultrafree spin concentrator and further concentrated to 60 µl. The concentrated protein sample was used for crystallization. BIOCHEMICA · No. 4 · 2001 27 Partially Purified PSP from Material in Lane 5 E. coli Lysate (RTS 500 E. coli HY Kit) PSP after Incubation at 30 °C for 20 hours Partially Purified PSP from Material in Lane 3 E. coli Lysate (RTS 500 E. coli Circular DNA Template Kit) Molecular Weight Marker Seite 28 PSP after Incubation at 30 °C for 20 hours M.W (kD) 16:17 Uhr E. coli Lysate prior to Incubation 24.09.2001 Molecular Weight Marker bc04-11-20.9.rz 50 40 30 20 10 " Figure 1: Coomassie-stained 15 % SDS-PAGE showing PSP produced in the RTS. Approximately 1 µl of reaction material was loaded in each lane RTS Crystallization and data collection PSP protein generated in the RTS was crystallized using the hanging drop vapor diffusion method with seeding in a buffer that had been utilized previously for crystallization of PSP produced in E. coli [1]. One µl of concentrated PSP was mixed with 1 µl of a well solution containing 0.1 M sodium acetate buffer, pH 4.5, 0.2 M sodium phosphate dihydrate, 5 mM MgCl2, and 22 % polyethylene glycol 2000 monomethylether (PEG2K MME). Micro-seeding was performed 1 hour after the drop was set up. Crystals appeared within 24 hours. The concentration of PEG2K MME was then raised to 30 % to stabilize the crystals. Crystals from the drop were flash frozen in liquid nitrogen and used directly for cryo-crystallography data collection. X-ray diffraction data were collected at the Advanced Light Source (ALS) (Berkeley, CA) beam line 5.0.2 using an Area Detector System Co. Quantum 4 CCD detector placed 140 mm from the crystal. 28 “Uniformly” 15N-labeled PSP An almost uniformly 15N-labeled PSP sample was generated by using 15N-algal amino acids (Cambridge Isotope Labs) with the RTS 500 E. coli HY Kit (Roche Molecular Biochemicals). A stock solution of 15N-labeled amino acids was prepared by dissolving 100 mg of the algal amino acid mixture in 6 ml of reconstitution buffer. Because the algal amino acid mixture is produced from acid hydrolysis of algal proteins, it does not contain Asn, Cys, Gln, or Trp. Except for Trp, which does not occur in PSP, 42 mM solutions of the unlabeled forms of the other three amino acids were used to supplement the 15N-algal amino acids. A total of 2.805 ml of 15N-algal amino acids stock solution was combined with 135 µl of Asn, 30 µl of Cys, and 30 µl of Gln to give a final volume of 3.0 ml. This labeling stock solution of amino acids was used together with the RTS 500 E. coli HY Kit according to the product instructions. The same parameters as described above were followed for protein production and purification, except for the concentration step. After the Hightrap-Q column, the fractions were pooled and the buffer was exchanged with a buffer containing 10 mM sodium phosphate at pH 6.5, 10 mM DTT, 20 mM MgCl2, and 0.5 mM EDTA. The protein was concentrated to a final volume of 450 µl. Fifty µl of D2O were added to the sample and the pH was adjusted to 6.5 prior to NMR data collection. 15N-Gly labeled PSP A 15N-Gly-labeled PSP sample was produced with the RTS 500 E. coli HY Kit by substituting unlabeled Gly with 15N-Gly (Isotech). The labeling stock solution was prepared by combining different amounts of 42 mM " Figure 2: Crystals of PSP generated with protein isolated from a 1-ml " Figure 3: X-ray diffraction data collected with the reaction using RTS 500 E. coli HY Kit. A) Picture of the entire crystallization crystal in Figure 2B. The crystal diffracted to a resolution drop. B) Close-up view of a crystal suitable for X-ray diffraction with limit of 1.5 Å, comparable to the resolution limit obtained dimensions of 100x150x100 µm with crystals of PSP expressed in E. coli BIOCHEMICA · No. 4 · 2001 ROCHE MOLECULAR BIOCHEMICALS http://biochem.roche.com bc04-11-20.9.rz 24.09.2001 16:17 Uhr Seite 29 102 102 106 106 110 110 114 114 118 122 126 126 130 130 134 134 138 138 10 9 8 10 7 9 8 1H-15N labeled with 15N-algal HSQC spectrum of PSP “uniformly” amino acids using the RTS 500 E. coli HY Kit. The spectrum was recorded on a Bruker 7 HN ppm HN ppm " Figure 4: N ppm 122 N ppm 118 " Figure 5: 1H-15N labeled with 15N-Gly HSQC spectrum of PSP selectively using the RTS500 E. coli HY Kit. The spectrum was recorded on a Bruker DRX 500 MHz AMX 600 MHz spectrometer with the sample temperature spectrometer with the sample temperature set to 25 °C set to 25 °C and a total recording time of 17 hours. Peaks and total recording time of 17 hours. Peaks corresponding corresponding to Asn, Cys, Gln, and Glu residues are to 14 of the 15 Gly residues in PSP were observed missing from the spectrum because the unlabeled forms of these amino acids were used to supplement the amino acid mixture RTS 15N-algal stock solution of each amino acid depending on the frequencies of occurrence of these amino acids in PSP. The volumes in microliter and copy numbers (in brackets) of each amino acid were as follows: 240 Ala (17), 105 Arg (8), 135 Asn (9), 195 Asp (13), 30 Cys (2), 30 Gln (1), 375 Glu (27), 225 15N-Gly (15), 0 His (0), 300 Ile (22), 270 Leu (20), 450 Lys (33), 45 Met (3), 105 Phe (7), 45 Pro (3), 75 Ser (5), 105 Thr (8), 0 Trp(0), 45 Tyr (3), 225 Val (15). The final volume of the amino acid mixture was 3.0 ml. The labeling stock solution was used according to the product instructions for the RTS 500 E. coli HY Kit. NMR data collection 1H-15N HSQC spectra [2] were recorded on Bruker AMX 600 MHz and DRX 500 MHz spectrometers. For each experiment, the sample temperature was set to 25 °C and the total recording time was 17 hours. The data were processed using the NMRPipe software suite [3]. (Figure 3). 1H-15N HSQC spectra of 15N-“uniformly” labeled and 15N-Gly selectively labeled samples of PSP generated in the RTS show encouraging results for NMR structure determination (Figures 4 and 5). Except for the weakest peak, all of the peaks in the 15N-Gly spectrum (Figure 5) can be superimposed with the corresponding peaks in the spectrum of the 15N-“uniformly” labeled sample (Figure 4). If similar results are obtained for a significant number of target proteins, in-vitro protein production with the RTS should play a major role in structural genomics as well as in structural biology in general. The authors would like to acknowledge Rosalyn Kim for performing the activity assays of PSP generated in the RTS System. References 1. Wang W et al. (2001), Structure Fold Design 9: 65-72. 2. Mori S et al. (1995), J Mag Reson B 108(1): 94-98. 3. Delaglio F et al. (1995), J Biomol NMR Nov, 6(3): 277-93. Results and Discussion RTS reactions were easy to set up and produced PSP consistently in each reaction. A 1-ml reaction with the RTS 500 E. coli HY Kit yielded 2–3 mg of protein in the crude reaction mixture and about 1.5 mg of purified PSP (Figure 1). In addition, PSP protein produced in RTS was active and could be crystallized using the same conditions for PSP expressed in E. coli [1] (Figure 2). X-ray crystallography data collected with synchrotron radiation at the ALS indicated that the crystals produced were of high quality, diffracting to a resolution limit of 1.5 Å ROCHE MOLECULAR BIOCHEMICALS http://biochem.roche.com http://www.proteinexpression.com Product Pack Size Cat. No. RTS 500 Instrument 1 instrument 3 064 859 RTS 500 E. coli HY Kit 1 kit (2 reactions) 1 kit (5 reactions) 3 246 817 1 kit (for 5 reactions) 3 262 154 RTS 500 Amino Acid Sampler 3 246 949 BIOCHEMICA · No. 4 · 2001 29