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Electrokinetic Isolation of Vesicles and Ribosomes Derived from Serratia marcescens Debra T. L. Hawker, Paul Todd, Robert H. Davis,*Robert C. Lawson,?and Scott R. Rudgei Department of Chemical Engineering, University of Colorado, Boulder, Colorado 80309-0424
Ribosomes and vesicles derived from the bacterium Serratia marcescens were separated from each other and from solubles using density gradient electrophoresis. Transport relationships were used to determine the electrophoretic mobilities of the particles. The effects of convection, sedimentation and diffusion were found to be negligible. The cm2/(V-s). Under electrophoretic mobility obtained for the ribosome peak is -7 X appropriate conditions, two vesicle peaks were obtained, the first with a mobility of -4 X cm2/(V-s)and the second with -9 X lo+ cm2/(V-s). This information can be used t o predict the resolution of the separands in large-scale electrophoretic separations.
1. Introduction Separation processes tend to be the most expensive and most time-consuming steps in the production of biotechnology products (Belter et al., 1988; Knight, 1989). Many separation methods have been studied and characterized for the preparation of soluble products such as proteins and large particulate products such as cells. However, there have been comparatively few studies concerning separation processes required to purify intermediate-sized products such as ribosomes and vesicles. Research was therefore undertaken to examine zone electrophoresis for the separation of a model system consisting of ribosomes and vesicles derived from the Gram-negative bacterium Serratia marcexem. Preparation of ribosomes and vesicles used in this study traditionally occurs in a series of lysis and centrifugation steps, including ultracentrifugation (Warren et al., 1989). The volume which can be processed in a single batch at the required rotor speeds is on the order of 2 L or less in several hours. Ultracentrifugation is limited to small volumes since it is typically a batch process and because the achievable centrifugal force decreases with increasing centrifuge size (Perry and Chilton, 1973). Thus, for large scale production of intermediate-sizedbiological products such as vesicles and ribosomes, other separation techniques need to be investigated. As commonly applied in biochemistry or biotechnology, zone electrophoresis effects the separation of separands that have different electrophoretic mobilities. The electrophoretic mobility determines the velocity with which a particle or molecule moves through a specified medium when an electric field is applied, and it is a function of the ionic strength of the medium, the {potential of the particle, the viscosity of the medium, and the shape of the particle. Here, it was desired to separate the ribosomes and the vesicles from the solubles remaining in a partially cleared cell lysate and from each other. If this could be accomplished efficiently using zone electrophoresis, it would result in the elimination of the final ultracentrifugation steps in the existing separation strategy. In the absence of other effects, particles with the same electrophoretic mobility will move at the same rate in a
* To whom correspondenceshould be addressed. + Present address: f
NaPro, 4880 SterlingDr., Boulder, CO 80301. Present address: Synergen,188533rdStreet,Boulder,CO 80301. 8756-7938/92/3008-0429$03.00/0
specified electric field. However, sources of motion in addition to the electric field may act on the particles. These include gravity, diffusion, free convection (driven by heat dissipation in the system), and electroosmosis (Snyder et al., 1986; Todd, 1990). The net result of the presence of these additional forces and flows is the spreading of the sample band and thus a reduction in resolution of the separands. Previous researchers, however, have overcome many of these difficulties in large-scale systems (Gobie et al., 1985; Mosher et al., 1987; Ivory, 1988). These systems typically control the fluid flow, thereby controlling the positions of the separands and overcoming free convection. Theoretical models exist that describe the separation capabilities of some of these apparatuses. Thus, the resolution between two or more separands can be predicted given the electrophoretic mobilities or {potentials of the separands and other pertinent data such as sedimentation velocity and diffusivity (Gobie and Ivory, 1988; Ivory, 1988). Density gradient zone electrophoresis (DGZE) is one method of obtaining electrophoretic data by using a relatively simple apparatus. In DGZE, a vertical density gradient is formed in the region where electrophoresis is performed, in order to stabilize the fluid column against free convection. The DGZE technique has been applied by previous researchers to various types of living and fixed cell separations (Boltz and Todd, 1979; Plank et al., 1983; Tulp, 1984). Sedimentation contributes greatly to the motion of these cells; in fact, two classes of pituitary cells were separated by sedimentation rather than by electrophoresis in DGZE experiments. A theory for the motion of the cells, including electrophoretic and sedimentation effects, has been presented and is in good agreement with the experimental results (Plank et al., 1981). Joseleau-Petit and Kepes studied the effect of DGZE on vesicles formed from Escherichia coli (Joseleau-Petit and Kepes, 1975). Their study is of particular interest, since the vesicles were prepared using a method similar to that used for preparing the materials chosen for our study. Their preparations presumably included ribosomes, and three peaks were apparent in the electrophoretic profile, with the first two correspondingto vesicles while the last appeared to contain ribosomes. Unfortunately, the data presented were not sufficient to calculate the electrophoretic mobilities of their fractions. The research presented below extends their
0 1992 American Chemical Society and American Institute of Chemical Engineers
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Figure 1. Density gradient apparatus: (A) 60% sucrose floor; (B) 0 4 5 % sucrose gradient; (C) 0% sucrose top solution; (D) three-waystopcock;(E)electrodetop solution;(F)saturated NaCI; (G) 15% polyacrylamide gel plugs; (H)cooling jacket.
results to the isolation of both ribosomes and vesicles derived from Serratia marcescem, and calculations of the electrophoretic mobilities are made.
2. Materials and Methods 2.1. Electrophoretic Apparatus. The density gradient electrophoretic apparatus used, as described previously, is shown in Figure 1(Boltz and Todd, 1979). The apparatus consists of a thermostated density gradient column connected to two glass electrode side arms via two Teflon blocks. The electrochemical reactions occur in the side arms without disturbing the sample or the buffer solutions within the density gradient column. The electrochemical reactions occur in the saturated sodium chloride solution (F),while the top solution (E) consists of a buffer which has an ionic composition similar to that within the density gradient and allows conduction of ions while the density gradient is protected from changes in ionic composition or pH. Polyacrylamide gel plugs (G) provide a physical barrier to fluid flow while allowing the conduction of ions into and out of the density gradient. Semipermeable membranes were used instead of gel plugs in some of the experiments. These membranes are easier to install and have a longer lifetime but do not otherwise affect the experiments (K. D. Cole, personal communication). The entrance and exit of the column are preceded by parabolic reducers which compress streamlines and minimize the effects of pumping the solutions into and out of the column. Electrophoresis occurs within the density gradient (B) which stabilizes the system against convection during field application and during pumping into or out of the density gradient column. All solutions are added to the column through the three-way stopcock (D). The ceiling (C)and the floor (A) solutions stabilize the position of the density gradient and are in electrical contact with the polyacryl-
amide gel plugs or the semipermeable membranes. The column coolingjacket maintains the desired temperature at the column wall and provides a sink for the heat generated by the flow of current in the column interior. During electrophoresis runs, the jacket was maintained at 4 OC. The density gradient columns used have an internal radius of R = 1.1cm and a gradient length of approximately 8 cm or 13 cm, depending on the run conditions; longer runs at higher currents required the longer column. 2.2. Buffer and Gradient Systems. The buffer employed consisted of 4 mM trizmabase (obtained from Sigma Chemical Co.), 10 mM MgSOr, 10 mM NH&l, and 50 mM glycine. The buffer was titrated to a pH of 8.2 at 4 "C with 6 M hydrochloric acid. A magnesium source was included in the buffer in order to stabilize the ribosomes (Urban, 1990). Glycine was added to the system to aid in pH control. The density gradient was based on sucrose. The top solution and the top of the gradient were sucrose-free for both columns, resulting in a density of p = 1.000 g/cm3 at 4 "C. The bottom of the gradient was 15% by weight sucrose ( p = 1.073 g/cm3)for the 8-cm column and 25% by weight sucrose ( p = 1.114 g/cm3)for the 13-cm column. The resulting density profiles along the column length were linear. The pumping solutions were 40% and 60% sucrose by weight, respectively. Conductivity and viscosity measurements were completed as follows. Linear combinations of the top and bottom solutions were assembled, and their properties were measured using a conductivity meter and a parallel disk viscometer. These data were compared to those from samples obtained from the column at appropriate positions, and no discrepancies were observed. 2.3. Sample Preparation. Cells were grown in batch fermentation, harvested, and then cooled before they were lysed in a French press (Urban, 1987). Lysis frees the ribosomes from the cells and causes the cell membranes to reform into vesicles which are much smaller than the parent cell. In addition, other cell fragments are created, and intracellular materials are released into the solution. The cell lysate was then centrifuged to remove the larger debris, in particular the cell and cell-wall fragments. The remaining partially cleared cell lysate then normally undergoes a series of ultracentrifugation steps to sediment the desired ribosomes and vesicles, leaving the undesired solubles in the supernatant. The average diameters of the ribosomes and vesicles are 25 nm and 250 nm, respectively (as measured by light scattering),and the wet densities are 1.20 and 1.22 g/cm3, respectively (as measured by isopycnic sedimentation). Four types of samples were studied by electrophoresis: partially cleared cell lysate, purified ribosomes, purified vesicles, and a mixture of the purified ribosomes and vesicles. All of the samples were kept on ice during the following procedures required to prepare the samples for electrophoresis. The density of the sample was determined using a densimeter, and the proper amount of sucrose required for the sample to sit on the density shelf (between the floor and the bottom of the densitygradient) was added to the sample, and the sample was mixed gently until the sucrose dissolved. The sample was then loaded onto the column and was checked to determine that the sample zone remained stable on the density shelf. Typical sample sizes were 0.10-2.0 mL (at 2% particulates by weight), which correspond to 0.2-4.0 mg of particulates. Occasionally 200 pL of saturated aqueous bromophenol blue solution was added to the sample to aid in determining fluid and pH stabilities. 2.4. Electrophoresis Procedures. The sidearms were prepared for assembly either by filling the bridges with
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15% polyacrylamide gel plugs (Boltz and Todd, 1979) or by cementing dialysis membranes (10 000-MW cutoff) to a perforated plastic septum which was inserted into the side arm and the Teflon blocks. The apparatus was then assembled as shown in Figure 1. When polyacrylamide gel plugs were used, it was necessary to purge them of contamination prior to beginning an electrophoresis run. This was done by loading the side arms and the density gradient column with distilled water, completing all electrical connections, applying an electric field overnight, and removing the water the following morning. Once the sidearms were purged, they were loaded with the appropriate solutions. First, the top buffer solution, with 0.5 M glycine added, was poured into the side arm. Saturated sodium chloride was then added to the side arm slowly through the stopcock at the bottom of the side arm. This allowed for the formation of two separate liquid layers within the side arm. At this point, the refrigerated cooler which feeds the water jacket was switched on and the system was allowed to equilibrate to 4 OC. Loading of the column proceeded as follows, with all solutions loaded through the stopcock a t the bottom of the column. First, 30 mL of the top solution was added to form the ceiling, then 30 mL of the density gradient, formed in a paired-cylinder linear gradient former, was added to form the 8-cm density gradient column or 50 mL was added to form the 13-cm density gradient column. The sample was then added, beneath the gradient, with a syringe at the stopcock. Care was taken to not disturb the gradient while the sample was injected. The floor solution of approximately 35 mL was then added until the sample became visible in the jacketed portion of the density gradient column. Typical electrophoretic separationswere conducted with the power supply in the constant current mode at 9-30 mA for 2-20 h. After separation, fractions were collected by pumping the pumping solution into the bottom of the column at a rate of 1 mL/min and collecting fractions off the top of the column using a fraction collector. The first 30 mL of solution removed was discarded, as this corresponds to the ceiling, and the next 30 mL was collected for analysis. Fractions of 0.5-2.0 mL were collected and analyzed for their ribosome, vesicle, and solubles content. The ceiling and floor fractions were also analyzed in early experiments to confirm that the sample was not migrating to the ceiling or the floor. 2.5. Analysis of Fractions. Optical density measurements were used to trace the ribosomes, vesicles, and solubles (Brown, 1980). The ratio of the optical density readings at 260 nm to that at 280 nm is approximately 1.9 for ribosomes and is approximately 1.4 for vesicles derived from Serratia marcescens. Samples were diluted with buffer (20 mM trizmabase, 20 mM MgS04,50 mM NHdC1, pH = 7.6 at 4 "C) to concentrations within the linear range of the spectrophotometer (less than 1.75 optical density units). Optical density measurements were made on samples in quartz cuvettes using a Hewlett-Packard 8452 series spectrophotometer at 260 nm and 280 nm. Sample purity was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) (Laemmli, 1970). After electrophoresis, the gels were stained with colloidal Coomassie Brilliant Blue to visualize the proteins (Neuhoff, 1988). The proteins of ribosomes and vesicles have specific banding patterns on SDS-PAGE gels, and the purity of the fractions was determined qualitatively by comparing the banding patterns of the fractions with that of particles of known purity.
431
2' 2.04
't? 1l.B
3
i i
.. 1.24 0
2
6
4
1.6
11.4 8
x (cm)
Figure 2. Conductivityandviscosityprofiles for the 8-cmdensity gradient. The symbols are measurementsfrom samplefractions, and the curves are the fits provided by eqs 3 and 4.
3. Data Analysis The data analysis assumes that convection, sedimentation, diffusion, electroosmosis, and other factors besides electrophoresis have a negligible effect on particle motion. Under these conditions, velocity of a nonconducting particle in an electric field is given by u=G=&i
dt k , where X is the vertical distance traveled by the particle in time t, 1.1is the electrophoretic mobility of the particle, j is the current flux, and ke is the electrical conductivity of the solution. The electrophoretic mobility is described by
where €0 = 8.85 X lo-' (g-cm)/(s2*V2)is the electrical permitivity of free space, D = 80 is the dielectric constant of the fluid, f is the zeta potential of the particle, g(Ka) is a constant (see below), and q is the viscosity of the solution. The conductivity and viscosity of the fluid are functions of the position within the density gradient column because of the presence of the sucrose gradient, whereas the variation of the dielectric constant is relatively small. The variation of the conductivity and viscosity are described empirically by
k , = ko(l + a , x ) q = T)o(l+
a2x
+ a3x2)
(3) (4)
where x is the vertical distance from the column floor and the coefficients are determined from least-squares regression of property measurements for several different fractions along the column. For the 8-cm column, ko = 1.38 mA/(V.cm), a1 = 0.057 cm-', 70= 0.0258 g/(cm.s), a2 = -0.072 cm-l, and a 3 = 0.0027 cm-2, whereas for the 13cm column, ko = 0.940 mA/(V-cm),a1 = 0.086 cm-I, QO = 0.0361 g/(cms), a2 = -0.074 cm-I, and a3 = 0.0024 cm-2. The viscosity and conductivity profiles for the 8 c m column are shown in Figure 2. The quantity Ka is the product of the Debye-Htickel constant (reciprocal of the double-layer thickness) and the particle radius. Huckel (1924) assumed that the electric field was not deformed by the particle, which corresponds to Ka
