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ANALYTICAL BIOCHEMISTRY ARTICLE NO.

238, 50–53 (1996)

0249

Upside-Down Stopped-Flow Electrofractionation of Complex Protein Mixtures Stanislav N. Naryzhny B. P. Konstantinov Institute of Nuclear Physics, Russian Academy of Sciences, Gatchina, Leningrad District, 188350, Russia

Received November 3, 1995

The excellent resolution of SDS–PAGE in protein analysis stimulated the creation of various preparative devices. The main approach used in these devices is the construction of a elution chamber in the lower end of the polyacrylamide gel cylinder or plate. Although this continuous lower buffer flow electrofractionation system serves as an acceptable preparative electrophoresis, some limitations to this approach exist. There is strong dilution of protein zones by the eluting buffer, which drastically restricts the sensitivity of the determination of minor proteins, and the restricted current flow caused by electric resistance arising from the column holder prevents application to purification of complex protein mixtures. To overcome these problems, the upside-down stopped-flow electrofractionation system (UDSFE) was designed. The necessary quantity of fraction is drawn with a pipet in a small volume from just above the gel cylinder. This invention improves the possibility of electrofractionation of deluted complex protein mixtures. The efficiency of this technique is demonstrated by purification a protein kinase from rat liver. The method has also been successfully used for purification of errorcorrecting 3*–5* exonuclease. q 1996 Academic Press, Inc.

The use of SDS–PAGE1 as a preparative method depends on the elution of separated proteins from a gel. This is achieved either by continuous or stopped-flow elution during electrophoresis or by slicing the gel after electrophoresis and subsequently eluting the protein zones of interest. The first approach is advantageous because of its more practical and quantitative possibilities. Therefore, many attempts have been made to design continuous elution devices suitable for routine pro1 Abbreviations used: UDSFE, upside-down stopped-flow electrofractionation; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; 2-ME, 2-mercaptoethanol.

tein purification, in which bands emerging from the bottom of electrophoresis gels are swept away to a fraction collector (1–3). Although the efficiency of this technique has been reported as being very high, some problems still exist. Among these are the high degree of protein zone dilution and the restricted current flow in the system that makes purification of minor components, such as eukaryotic enzymes, from complex protein mixtures more difficult. The stopped-flow approach has the same problems and has proved impractical so far (4, 5). To avoid these problems and to be able to work without a column holder, the so-called upsidedown stopped-flow electrofractionation (UDSFE) was designed. The patent approach is extremely simple and possible for every laboratory. In this report, I describe the use of the UDSFE system to purify a protein kinase from rat liver. The important role of this class of enzymes in the regulation of a number of cell processes including DNA replication has been successfully clarified during the past decade (6–8). MATERIALS AND METHODS

Partial Isolation of Protein Kinase Male albino rat liver (3.5 g) was homogenized in a Dounce homogenizer at 47C in 40 ml medium: 0.25 M sucrose, 1 mM EDTA, 3 mM MgCl2 , 50 mM Tris–HCl buffer (pH 7.4), 2 mM 2-mercaptoethanol (2-ME) (Ferak, Germany). The homogenate was filtered through eight layers of gauze, and the cytosol was separated from the nuclei by centrifugation at 6000g for 10 min. The complex form of protein kinase with DNA polymerase a was sequentially isolated from the cytosol by ion-exchange chromatography on DEAE–Sephadex (Pharmacia-LKB), where the active fraction was eluted at 0.2 M NaCl. It was precipitated by 40% saturated ammonium sulfate and purified on Sephacryl S-300 (Pharmacia-LKB). Three protein kinases were eluted, one of them together with DNA polymerase a in the 400-kDa protein region. The columns were equilibrated

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FIG. 1. Schematic view of the UDSFE system. (1) The initial position. A sample is embedded upside-down in the gel and electrophoresis is carried out from the top down. (2) The second position. When a bromphenol blue front reaches the resolving gel end, the glass tube is turned upside-down. (A) Glass tube with resolving and stacking gel; (B) electrode reservoirs; (C) buffer level, when the buffer forcingout unit is down; (D) buffer level, when the buffer forcing-out unit is up; (E) section in which proteins are eluted; (F) direction of electromigration; (G) buffer forcing-out unit (a round hole in the center).

and eluted with 50 mM Tris–HCl, pH 7.5, 5 mM 2-ME, 2 mM EDTA, 10 mM Na2S2O5 , 5 mM phenylmethylsulfonyl fluoride. Protein Kinase Activity Determination Protein kinase activity was assayed by the rate of terminal phosphate transfer from [g-32P]ATP to BSA. The assay mixture (100 ml) contained 50 mM Tris– buffer, (pH 7.5), 20 mM MgCl2 , 2 mM 2-ME, 0.5 mg/ ml BSA, 5 mCi/ml [g-32P]ATP (1 mCi/mmol), and the enzyme. Assays were carried out at 377C and terminated by the addition of 5 ml of cold 10% trichloroacetic acid. A precipitated protein was collected on a Synpor3 filter (Reanal, Hungary), washed with ethanol, and dried. Dried filters were counted in a toluene scintillation mixture on a Beckman Model LS 5801 scintillation counter. The activity of DNA polymerase a was determined by the rate of [3H]TTP incorporation into acid-insoluble DNA (9). Protein was determined according to Bradford (10). Acrylamide, bisacrylamide, SDS, aldolase, BSA, catalase, ovalbumin, and horse heart myoglobin were from Serva. Liquid chromatography was performed using the Pharmacia-LKB FPLC system. Standard analytical SDS–PAGE was performed in 1-mm polyacrylamide slab gels (10%) according to Laemmli (11). After electrophoresis, gels were stained with Coomassie R250 or silver (12).

retic column, a 90-mm-length glass tube with an outer diameter of 15 mm and an internal diameter of 13 mm was used. To prepare a gel, the lower side of the glass tube was sealed with parafilm, and 250 ml of 50% glycerol was poured on the bottom. Resolving and stacking gels were formed with 75 and 3 mm, respectively, above this viscous solution. The resolving gel concentration in these experiments was 10%. After polymerization, the tube was installed into a device through a hole in the partition between anode and cathode reservoirs. Hermeticity was achieved by putting a rubber ring outside the glass tube. After this, the standard electrophoresis buffer (50 mM Tris, 150 mM glycine, 0.1% SDS, pH 8.3) was poured into both reservoirs. The capacity of each was 400 ml. The parafilm was removed from the tube end, and a vacant 250-ml volume (E) was formed in place of the 50% glycerol. Then, 1000 ml of a sample (100–1000 mg of protein) was loaded onto the gel. Electrophoresis was carried out at room temperature at 80 V until the electrophoresis front (bromphenol blue) reached the lower edge of the resolving gel (position 1, Fig. 1). The power was turned off, the instrument was turned over, and the electrophoresis buffer in the anode reservoir was replaced with the same buffer without SDS but containing 0.2 M NaCl, 10% glycerol, and 1 mM 2-ME (high molarity salt buffer). Also, a forcingout unit (G) was placed in such a manner that the liquid level was a bit higher than the tube edge. Thus, if this unit is lifted, the liquid level will fall below the tube edge, and the buffer in section E will be separated from the main reservoir (position 2, Fig. 1). Just above the gel surface a zone of high voltage was formed by injection of 100 ml of low salt buffer (40% glycerol, 10 mM Tris, 30 mM glycin, 1 mM 2-ME, pH 8.3). The power was switched on again and electrophoresis was continued at 100 V. Now protein zones were migrating from

Instrumentation for Electrofractionation The schematic and general view of the electrophoresis system are shown in Figs. 1 and 2. As an electropho-

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FIG. 2. A general view of the UDSFE system.

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STANISLAV N. NARYZHNY

FIG. 3. Electrofractionation results showing distribution of protein zones and protein–kinase activity in the eluted fractions. One thousand microliters (300 mg) of partly purificated protein kinase from rat liver cytosol (see Materials and Methods) was loaded onto the gel. (A) SDS–PAGE of fractions eluted in the UDSFE system. Fifty microliters of each eluted fraction was analyzed by standard SDS–PAGE in a slab gel. Staining by silver. M, molecular mass markers in kDa; subunit of aldolase (40) and subunit of catalase (60). 0, a sample before fractionation. (B) Determination of protein kinase activity in eluted fractions. Just after electrophoresis 50 ml of each fraction was taken and cooled at 47C, SDS was precipitated, and the supernatant was placed in a protein kinase determination mixture (see Materials and Methods) and incubated for 30 h at 377C.

bottom to top and gathering in section E in the interphase between the low and the high molarity salt buffers because of their slow mobility in a high molarity salt buffer. The buffer level in the upper reservoir was lowered every 5–10 min by lifting the forcing-out unit, and the eluted proteins were drawn with a pipet from section E in a 250-ml volume. All these operations were performed by means of a special patented instrument which greatly facilitated all the above-described manipulations, but could be performed with standard laboratory equipment. RESULTS

UDSFE was used to purify protein kinase from rat liver cytosol. One thousand microliters (300 mg) of partially purified protein kinase (see Materials and Methods) was loaded onto the electrophoretic column. The specimen was heated at 507C for only 2 min to avoid irreversible protein denaturation. It takes 150 min for the electrophoresis front (bromphenol blue) to reach the gel edge at 80 V. After that, the power was turned off, the instrument was turned over, and the anode buffer was changed. The power was turned on and electrophoresis was carried out from bottom to top under 100 V. Every 10 min or more frequently, fractions of 250 ml were collected beginning from the bromphenol blue fraction (fraction N1). For enzyme activity determination, 50 ml was taken from each fraction. SDS was precipitated by cooling to 47C; the supernatant, containing the main protein quantity, was placed in a protein kinase activity determination mixture (see Materials and Methods) and incubated at 377C. For analytical SDS–PAGE, 50 ml from each fraction per line was taken without cooling.

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As Fig. 3A shows, proteins were collected according to their molecular weights. The resolution of this technique was about 2 kDa when proteins from 40 to 60 kDa were eluted and collected every 10 min. The resolution is increased with increasing protein weight. By adjusting the fractionation time, the desired resolution level can be obtained. Moreover, the protein concentration in separated fractions was higher than that in the initial specimen due to the small volume of collected fractions. Every fraction of 250 ml contained about one polypeptide which initially was 1000 ml in volume. This happened because the yield of this technique is about 90–95%. These measurements were carried out by densitometry of the analytical electrophoregram on which specimens of marker proteins (aldolase, ovalbumin, BSA) before and after fractionation were loaded (not shown). As shown in Fig. 3B protein kinase activity coincided with the polypeptyde of about 60 kDa. For further analysis some enzyme-containing fractions from different fractionations were combined and dialyzed for complete SDS removal. Protein kinase activity was better restored under these conditions. The yield of activity in this case was about 1%, and specific activity before and after fractionation was about 107 cpm/mg for 30 min at 377C. DISCUSSION

The detection and recovery of protein activities after SDS–PAGE are a desirable goals in the purification and analysis of proteins. This is particularly important with enzymes present in low amounts in the cell. Moreover, often these proteins are minor components of large, multienzyme complexes, and the usual chro-

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matographic purification procedures are unsuccessful. Due to its high resolution, SDS–PAGE is the most popular method for analysis of protein mixtures, although the presence of SDS frequently impedes determination of biological activity after electrophoresis. Therefore, sensitivity of detection of enzyme activity in eluted fractions after SDS–PAGE is the main problem in enzyme purification. Although the continuous lower buffer flow electrofractionation system serves as an acceptable preparative electrophoresis and is commercially available, there are still some limitations. A strong dilution of protein zones by the eluting buffer drastically restricts the sensitivity of determination of minor proteins. The restricted current flow caused by electric resistance arising from the column holder prevents application to purification of complex protein mixtures. Also it is necessary to mention the high cost and complexity of commercial devices. The proposed approach greatly helps in resolving all of these problems. First, it has high resolution, at which levels as high as necessary can be obtained and which depend only on the electrophoresis resolution. One must choose only the desired gel concentration, length, and fractionation time. Second, the dilution of desired protein zones may be minimal, strongly enhancing the possibility of enzyme activity detection. Also, protein loss is minimal. Finally, the proposed instrument is extremely simple and has a low cost. Using this method I attempted to identify and purify one of the protein kinases from rat liver. This kinase is isolated together with DNA polymerase a and possibly takes part in the regulation of DNA replication. As indicated in Fig. 3, protein kinase activity is partially restored under these conditions. Although precipitation by cooling does not remove SDS completely, it significantly lowers its content. Further SDS delution by the kinase activity determination mixture has the possibility of protein renaturation. The addition of Triton X-100 did not improve these results, but helped markedly in the purification of another enzyme, an error-correcting 3*–5* exonuclease. Thus, one should consider such a quick SDS removal as an acceptable, direct micromethod for enzyme renaturation and identification, but not for isolation of an active enzyme. When the enzyme-containing fraction was dialyzed for complete SDS removal, the enzyme activity increased markedly. The above-described method and device provide an

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opportunity to obtain the necessary protein in preparative quantities sufficient for sequence determination or immunization and to accomplish the main task of reverse genetics—to go from a protein to its gene. It especially provides good possibilities for purification of proteins and enzymes present in cells in low amounts and for which the usual chromatographic purification procedures are unsuccessful because of strong aggregation. In particular, this applies to membrane proteins. With regard to classic enzymatic investigation of homogeneous proteins, some difficulties arise from high (100 times) enzyme inactivation after denaturation. Although it is now possible to be optimistic, keeping in mind the last results of enzyme reactivation by chaperions (13), this would allow one to better study enzyme characteristics. In conclusion, there are no reasons for not using the described method and device to fractionate other biomolecules, not only proteins, in various buffer systems and gels. ACKNOWLEDGMENTS The author thanks Professor V. Krutyakov for critically reading the manuscript. This work was supported in part by the Russian Fund of Fundamental Investigations (Project Code 95-04-11084) and ISF (Grant NJ9J100).

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