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inding optimal buffer conditions for protein purifica- tion and concentration is crucial for the success in the drug discovery process (High Throughput Screening and biophysical characterization).However, this step is often laborious and time consuming as there are a large number of buffer conditions to be examined. DLS techniques have proven to be invaluable for char- acterizing the homogeneity of proteins in solution. The recent appearance of the DynaPro-MSXTC Titan Plate Re- ader has drastically increased throughput. Further, the co- mbination of the DLS plate reader technology with a comp- uter assisted optimal experimental design helps in a more efficient way to set up a screen for the optimal buffer condi- tions and identify the factors (or interaction of factors) im- portant for protein homogeneity. This study illustrates how the optimal buffer condi- tions were identified with the help of optimally designed buffer screens conducted for “Protein X” on a DynaPro-MSX TC Plate Reader. The experimental design was made with MODDE 7 software from Umetrics. We chose a range of buffers com- monly used in protein purification and biophysical studies (TRIS pH 7.5; TRIS pH 8.5; HEPES pH 7; HEPES pH 8; Na phosphate pH 6; Na phosphate pH 8; Bicine pH 9 and am- monium acetate solution at pH 7.3). In addition, as a vari- able for each of the buffers, we set the ionic strength (0-200 mM NaCl), glycerol content (0-10 v/v %) and detergents at 0.2CMC (β-OG; TritonX-100; CHAPS; dodecyl maltoside). The program returned 72 different buffer conditions to be screened (compared to about 400 buffers in the case when one separate factor is changed at a time). The set of buffers included 4 identical ones for a check of reproducibility. All the buffers were filtered through 200 nm filters prior to use and gave a random correlation function when tested in the DLS plate reader. The buffers were loaded into the wells of a 384-well plate at 45 µl/well. Recombinant Protein X was obtained at concentration of 2 mg/ml directly after purification in 20 mM TRIS buffer pH 8, 150 mM NaCl. The stock solution of the protein was spun on a bench centrifuge for 5 minutes to remove possible large aggregates from the solution. Directly after that, 5µl of the protein was added to the wells containing the buffers (approx 10µg/well). Solutions were left for incubation for 1 h at 4ºC. In the buffer screen we took advantage of an Auto La- ser Power Adjust function available on the DynaPro-MSX TC Plate Reader. It provides the optimal measuring conditi- ons for every well in the screen. The final data on Protein X homogeneity were analysed with the help of Modde software. As a part of the buffer optimization process, a batch mode DynaPro-MSXTC instrument was used to check for the effect of the temperature on the aggregation state of the sample. 12µl of the protein solution was loaded into the cuvette and scanned from 4 to 50ºC. As shown in Fig. 1, most buffer conditions tested were unfavourable as large aggregates were detected in most of the wells. The reproducibility of the data was good. It appeared that in some cases Protein X solutions con- tained very large aggregates that were causing large fluctua- tions and occasional saturation of the detectors, even at the lowest values of the laser power. After a thorough exami- nation of the correlation curves obtained in the screen the relevant ones were fitted with a regularization function to obtain the estimates of the Protein X homogeneity under dif- ferent conditions. The Modde program was applied to iden- tify the major factors or combinations of factors important for the Protein X homogeneity. DynaPro Plate Reader for Buffer Optimization DAWN ® , miniDAWN ®, ASTRA ® , Optilab ® , DynaPro ® , Protein Solutions ® and the Wyatt Technology logo are registered trademarks of Wyatt Technology Corporation. ©2005 Wyatt Technology Corporation Light Scattering for the Masses™ We found that low salt content in combination with particular detergents (either β-OG or dodecyl maltoside) and the presence of glycerol were essential for the homogeneity of Protein X solutions. In two cases, protein solutions ap- peared to be rather homogeneous. This was confirmed with the batch instrument. The best buffers were 20 mM HEPES at pH 7 or 15 mM sodium phosphate buffer at pH 8.0. This note graciously submitted by Natalia Markova, Biovitrum AB, RET-A3, Stockholm, SWEDEN. 040921B:2_Fractions 040917B:2_280nm2A,IMP 0.00 0.05 0.10 0.15 0.20 0.25 AU 0.0 0.5 1.0 1.5 2.0 ml 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Figure 1. Overview of the results obtained with the DynaPro plate reader in the optimal buffer screen (approx. 10µg protein X /well) Figure 2. Results of the analytical gel filtration of protein X solutions:(blue line) in the optimal buffer chosen on the basis of the DLS plate reader screen; (red line) in a reference buffer. Figure 3. The results of a temperature scan of protein X solution in the best buffer from DLS buffer screen. Experiment carried out on DynaPro.
