1 Department of Internal Medicine I, Laboratory for Tumortherapy, University of Cologne, 50924 Cologne and 3 Aqua Research, 50974 Cologne, Germany
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Abstract |
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Keywords: affinity chromatography/chromatography system/expression conditions optimization/parallel protein purification/recombinant proteins
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Introduction |
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Materials and methods |
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An overview of the system is presented in Figure 1. A buffer reservoir designed to permit flow at identical flow rates to 24 circular arranged outlets is connected to 24 affinity columns. The flow is produced by gravity force and the flow rate is primarily controlled by the fluid head pressure. Adjustment of the flow rate is achieved by varying the height of the buffer fluid level above the affinity resin. The technical details are shown on the right.
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Purification procedure
Disposable 1 ml columns were filled with Ni-NTA affinity resin, connected to the tubings and equilibrated in running buffer (PBS, pH 7.4). Protein preparations were loaded manually on each column and the columns were reconnected with the tubings to the buffer reservoir. In some cases, a wash procedure was performed to decrease unspecifically bound protein prior to elution. To this end the respective buffers (PBS, pH 7.4, containing 0.5 M NaCl; PBS, pH 7.4, containing 10 mM imidazole) were filled into the reservoir and the columns were washed until the reservoir was almost drained. Then the clamps at the respective tubings were closed and the buffer reservoir was completely emptied and filled with the next wash buffer. This ensured that the tubings always stayed full of fluid and no air was introduced into the flow. After the last prewash step, columns were washed again with PBS (pH 7.4) and proteins were eluted manually with 50200 mM imidazole (Sigma-Aldrich, St. Louis, MO). The first 2 ml of elution fractions contained the specific protein. The fractions were pooled and dialyzed against PBS (pH 7.4) to remove the imidazole. The affinity resins were taken out of the columns and pooled for the regeneration procedure according to the manufacturers instructions.
Biochemical and functional testing of the purified proteins
Yields of specific proteins were measured as follows. Protein samples were subjected to scanning spectrophotometry (Figure 2A) and total protein amount was calculated from the absorption at 280 nm. Purity was assessed by SDSPAGE and subsequent staining with Coomassie Brilliant Blue. The specific protein yield was calculated from total amount of protein and the purity of the specific protein.
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FACS
Target cells were incubated with the fusion proteins at 10 µg/ml, detected with a FITC-labeled secondary antibody and measured on a flow cytometer (Becton Dickinson Biosciences).
Coagulation assay
Coagulation function was determined as described previously (Gottstein et al., 2001). Briefly, fusion proteins were incubated with a coagulation factor mix and factor X. The ability of the fusion protein to activate factor X to Xa was measured using a chromogenic substrate specific for factor Xa and measuring the increase in absorption at 405 nm.
[3H]Leucine incorporation assay
This assay measures the protein synthesis of immunotoxin-treated cells in comparison with untreated control cells. It was performed as described previously (Gottstein et al., 1994). Briefly, target cells were incubated with immunotoxins in triplicate at different concentrations, then 3H-labeled leucine was added and after 24 h cells were harvested on a glass-fiber filter. The radioactivity of the filter pieces was measured in a scintillation counter and the percentage of protein synthesis in comparison with protein synthesis of untreated control cells was calculated.
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Application results |
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In this experiment, a 60 kDa fusion protein containing a His-tagged scFv fused to a coagulation-inducing protein was expressed under varying expression conditions to identify the conditions for highest yield. Nine different conditions were tested in duplicate, i.e. 18 samples were processed. Proteins were extracted via osmotic shock as described previously (Gottstein et al., 2001) from 100 ml of Escherichia coli suspension each. The protein preparations were purified on the affinity chromatography system. Yields of specific protein ranged from 4 to 80 µg, allowing us to identify the best expression conditions regarding the yield of protein (Figure 2A
). Subsequent binding and coagulation assays confirmed that the proteins were functionally active.
Application 2. Expression of His-tagged immunotoxins and optimization of function
Recombinant immunotoxins containing a 6x His-tag were prepared as described previously (Derbyshire et al., 1997) and expressed under varyious conditions to identify the conditions resulting in the best functional activity. Twelve different samples were tested. Expression and purification of proteins were performed analogously to the procedure described in application 1. The concentration of the proteins was determined and a [3H]leucine incorporation assay was performed. Inhibition curves are shown in Figure 2B
. Inhibition of target cells at the highest concentration tested ranged from 25 to 55%.
In conclusion, we have described the construction of an affinity chromatography system that combines low cost and the possibility of purifying in a gentle process a large number of samples in parallel. This system is easy to build and allows the optimization of expression conditions of recombinant proteins. It is also suitable for purifying a number of mutation variants of a protein in order to compare their functional activities. We have used this system successfully to optimize expression conditions of various recombinant proteins.
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Notes |
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Acknowledgments |
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References |
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Gottstein,C., Schön,G., Tawadros,S., Kube,D., Wargalla-Plate,U.C., Hansmann,M.L., Wacker,H.H., Berthold,F., Diehl,V. and Engert,A. (1994) Cancer Res., 54, 61866193.[Abstract]
Gottstein,C., Wels,W., Ober,B. and Thorpe,P.E. (2001) BioTechniques, 30, 190200.[ISI][Medline]
Received April 5, 2002; revised July 25, 2002; accepted July 25, 2002.