Use of Flow Field-Flow Fractionation forthe Rapid Quantitation of Ribosome andRibosomal Subunits in Escherichia coli atDifferent Protein Production Conditions

Mikael Nilsson,1 Leif Bulow,2 Karl-Gustav Wahlund1

1 Technical Analytical Chemistry, Center for Chemistry and ChemicalEngineering, Lund University, P.O. Box 124, S-221 00 Lund, Swedentelephone: 1INT 46 46 222 83 16; fax: 1INT 46 46 222 45 25;e-mail: karl-gustav.wahlund@teknlk.lth.se

2 Pure and Applied Biochemistry, Center for Chemistry and ChemicalEngineering, Lund University, Lund, Sweden

Received 31 May 1996; accepted 25 October 1996

Abstract: Asymmetrical flow field-flow fractionation was and subunits due to the rather laborious experimentsused for rapid (8–14 min) separation of ribosomes and and the experimental skill required (Birnbaum andtheir subunits. The amount of ribosomes and the mass Bailey, 1991). Moreover, artifacts occurring during ul-fraction of ribosomes was determined in growing Esche-

tracentrifugation have been reported (Hauge, 1971;richia coli cells. These quantities changed significantlySpirin, 1971). These are connected to factors such asat different growth phases. Ribosomal composition was

monitored after the insertion of a protein-encoding plas- the long process time and the high hydrostatic pressure.mid and after the addition of an antibiotic agent. The Improvements in the analytical methodology can openresults suggest that the method will be useful in studies up new possibilities to study ribosomal systems.of, e.g., the relationships between the protein production

We have previously shown (Nilsson et al., 1996) thatcapacity of cells and the ribosomal composition. The anal-asymmetrical flow field-flow fractionation (asymmetri-ysis time is substantially shorter than ultracentrifugation

run times. 1977 John Wiley & Sons, Inc. Biotechnol Bioeng cal flow FFF) can be used to separate the ribosome54: 461–467, 1997. from the subunits and to quantitate the mass fractionKeywords: flow field-flow fractionation; ribosomes; sub- of ribosome in preparations of Escherichia coli. Theunits; quantitation

method fractionates colloidal particles based on differ-ences in their diffusion coefficients, which depend on

INTRODUCTION the hydrodynamic size. A fractionation of differentlysized components is thus achieved (Giddings, 1993). TheProteins are synthesized inside biological cells by theparticles are transported in a laminar flow by an aqueousribosomes, which translate the genetic code to proteins.carrier through an open channel and are at the sameThe ribosomes are composed of two subunits, and onlytime subjected to a flow of the carrier (a ‘‘crossflow’’)when these are combined to a complete ribosome doesin a direction perpendicular to the axial channel flow.protein synthesis occur. To understand the protein-The sample components are thus eluted through thesynthesizing machinery, a measurement of the activitychannel, and separated fractions can be collected. Theof the ribosomes is important (Jinks-Robertson and No-separation mechanism is based on the balance betweenmura, 1987; Maaløe, 1979).the crossflow-induced transport and the oppositely di-An important goal of metabolic engineering is the

maximization of the protein production capacity rected diffusional transport caused by Brownian motion.Thus the elution times for each separated componentachieved through optimization of the ribosomal system.

Knowledge of the number of ribosomes per cell and the can be directly converted into hydrodynamic diame-ters. Separations are often finished in 2–10 min. Thesemass fraction of the ‘‘active’’ ribosomes is then essential.

This requires measurements of these factors under a short separation times will give results which are morein vivo relevant than using ultracentrifugation tech-wide range of experimental conditions. Ultracentrifuga-

tion has been employed only in a limited number of niques.In the present report, we show the potential of theexperimental studies for the determination of ribosome

FFF method by analyzing the mass fraction of ribosomeas well as the number of ribosomes per cell at variousCorrespondence to: K.-G. Wahlundphases of an E. coli growth curve and after introductionContract grant sponsors: Swedish Natural Science Research Coun-

cil; Astra Hassle AB; Carl Trygger Foundation of a plasmid into a bacterial host. We have also studied

1997 John Wiley & Sons, Inc. CCC 0006-3592/97/050461-07

the effect on the ribosomes of the addition of chloram- and incubating 10 min on ice. The suspensions werethen exposed to a freeze–thaw cycle (liquid nitrogen–phenicol. The method should be particularly useful in

studies of the relationship between ribosomal composi- water at 208C) four times. Then 400 mL buffer A, 50 mL5% (w/v) Brij 58 in buffer A, and 3 mL RNAse-freetion and the protein production capacity of genetically

engineered cells whose growth conditions have been DNAse were added. The suspensions were incubated10 min on ice and centrifuged at 13,000 rpm for 3 minaltered in order to optimize protein production.in an Eppendorff centrifuge. The supernatants were

MATERIALS AND METHODS transferred to new tubes and stored at 48C awaitingFFF analysis.

Cultivation Conditions

In all cultivations the flasks were shaken at 130–140 Standard Samplesrevolutions per minute at 378C, and bacterial growth

Escherichia coli tRNA was purchased from Sigma. Thewas monitored by measuring the optical density atsubunits, prepared as previously described (Pettersson550 nm (OD550).et al., 1976), were a gift from A. Liljas, Molecular Bio-For the growth rate experiments 200 mL Luria–physics, Lund University.Bertani (LB) medium (1% bacto-tryptone, 0.5% NaCl,

and 0.5% yeast extract) in a 1000-mL flask was inocu-lated with 1 mL of an overnight culture of E. coli TG1. Field-Flow Fractionation AnalysisSamples were aseptically withdrawn at an OD550 of 0.11,

The instrumental setup was similar to that in our previ-10.3, 14.8, and 11.8.ous report (Nilsson et al., 1996) with some minor modi-For the experiments with chloramphenicol 50 mL LBfications. The sample injection pump was a CMA/100medium in a 250-mL flask was inoculated with 0.5 mLmicroinjection pump (CMA/Microdialysis AB, Stock-of an overnight culture of E. coli TG1. After 5.5 h ofholm, Sweden). The spectrophotometric diode array de-cultivation (OD550 5 3.5) a sample of bacteria was with-tector was an UVD 340S (Gynkotek, Germering, Ger-drawn and chloramphenicol was added to a final concen-many). Data acquisition and control of the two- andtration of 30 mg/mL. After an additional 4.1 h of cultiva-four-way valves and pumps were carried out with thetion (OD550 5 3.8) a sample was withdrawn.Gynkosoft software (Gynkotek, Germering, Germany).For the plasmid experiments two 250-mL flasks con-

The design of the asymmetrical flow FFF separationtaining 50 mL LB medium were inoculated with 0.5 mLchannel has been described before (Nilsson et al., 1996).of an overnight culture of E. coli TG1 harboring theDuring this work several channels were used. They allplasmid pUHL 1842 (Carlsson et al., 1993). Ampicillinhad a length, L, of 28.5 cm; breadth at inlet, bo, of 2.0was added to a final concentration of 100 mg/mL. Thecm; breadth of outlet, bL, of 0.50 cm; and area cut offplasmid was induced by adding isopropyl-thio-b-D-at the tapered inlet end, y, of 2.1 cm2 (see Fig. 1). Thegalactoside, to a final concentration of 0.2 mM to onethicknesses, w, of channels I through V were calibratedof the flasks after 3 h of cultivation (OD550 5 0.4). Afterwith ferritin (Litzen, 1993) and were 178, 185, 154, 143,an additional 2 h and 35 min one sample from each flaskand 180 mm, respectively. The flow rates were chosenwas withdrawn (OD550 5 3.3). As a control, the plasmid-so that the efficiency of each channel was optimized tofree strain E. coli TG1 was grown using identical condi-maximize the resolution between the large subunit andtions and a sample was withdrawn after 5 h and 25 minthe ribosome (Nilsson et al., 1996).at OD550 5 3.3.

The channels were equipped with an ultrafiltrationmembrane of regenerated cellulose, type Nadir UF-C-Cell Harvest10 (Hoechst AG, Wiesbaden, Germany), with a 10,000

The samples (13.0 or 15.0 mL) of the bacteria cultures molecular weight cutoff. The carrier liquid was bufferwere poured on 20–25 g crushed ice with 3.25 mL 1% A or, in the experiments at low magnesium ion concen-(w/v) NaN3. The suspensions were centrifuged for 5 tration, buffer A with MgSO4 decreased to 1 mM. Themin at 5900g at 48C. The pellets were resuspended in injected sample volume was 20 mL. The FFF analysis1 mL buffer A (25 mM Tris buffer with 10 mM MgSO4 was conducted and the data interpreted as describedand 30 mM K2SO4 , pH 7.4) and centrifuged 3 min at before (Nilsson et al., 1996).13,000 rpm in an Eppendorff centrifuge and then re-suspended in 1 mL sucrose buffer [buffer A with 18%

Electron Microscopy(w/v) sucrose]. The resuspensions were frozen in liquidnitrogen in 40-mL aliquots and stored at 2208C. Fractions of the three ribosomal peaks were collected

and analyzed by transmission electron microscopy. Ri-Preparation of Ribosomes bosomes and subunits were adsorbed to carbon film on

copper grids and subsequently negatively stained withRibosomes were prepared from the 40-mL aliquots byadding 10 mL buffer A containing lysozyme (5 mg/mL) 2% uranyl acetate and observed in a Philips CM 10

462 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 54, NO. 5, JUNE 5, 1997

Figure 1. Separation channel (top) and principle of separation (bottom). The breadth at the inlet and outlet is bo and bL, respectively. Thearea cut off at the inlet end is y. The length of the channel is L and the thickness is w. The axial laminar flow in the channel results in aparabolic velocity profile. The transversal position of the sample particles is determined by their Brownian motion and the crossflow velocity.Larger particles or molecules are less diffusive and end up closer to the wall where they are subjected to lower flow velocities in the parabolicprofile. They are thus eluted later than smaller particles which experience higher flow velocities.

electron microscope at 60 kV. From each negative 34–53 fractogram 1 of Figure 2 are the small subunit (30S),the large subunit (50S), and the ribosome (70S), in theparticles were selected and the average of two diameters

perpendicular to each other was calculated. mentioned order. Their hydrodynamic diameters were17, 20, and 24 nm, which is close to the known values(Nilsson et al., 1996).

Calculations Support for the identity of the three peaks was ob-tained by preparing and analyzing the ribosomes inThe retention time was translated into a hydrodynamic1 mM Mg21 (Fig. 2, fractogram 2) where they should bediameter as described previously (Nilsson et al., 1996;

Wittgren et al., 1996).The mass fraction of ribosome was estimated by divid-

ing the peak height at the maximum of the ribosomepeak by the sum of peak heights at the peak maximaof the two subunits and the ribosome peaks. The amountof ribosomes was estimated by multiplying the area un-der the ribosome peak (defined by constructing a base-line and drawing a line perpendicular to the baselinefrom the valley between the large subunit peak andthe ribosome peak) by the outlet flow rate (Vout) anddividing by the extinction coefficient for ribosomes at260 nm (4.2 3 1027 M21 cm21) and the detector pathlength (1 cm). The number of ribosomes per cell wasthen determined using Avogadro’s number and the cellnumber obtained from OD550 measurements (using acalibration constant of 2.0 3 108 cells/mL corresponding

Figure 2. Separation of ribosomes and subunits by flow FFF andto OD550 5 1). The calibration constant was obtainedidentification of peaks. Fractogram 1: preparation made with 10 mMby cell counts on serial dilutions at different phases ofMg21 in the preparation buffer and in the carrier liquid. Channel Ithe growth curves. was used. The outlet flow rate, Vout, was 0.44 mL/min and the crossflowrate, Vc, was 5.4 mL/min. The void time, t0, was 0.28 min. Fractogram2: preparation made with 1 mM Mg21 in the preparation buffer and

RESULTS AND DISCUSSION in the carrier liquid (channel II, Vout 5 1.1 mL/min, Vc 5 4.7 mL/min, and t0 5 0.21 min). Fractogram 3: purified large subunit (channelIV, Vout 5 0.53 mL/min, Vc 5 5.3 mL/min, and t0 5 0.22 min).

Identification of Ribosome and Subunits Fractogram 4: purified small subunit (channel IV, Vout 5 0.53 mL/min, Vc 5 5.4 mL/min, and t0 5 0.22 min). Fractogram 5: standard

Samples containing ribosomes from growing E. coli cells E. coli tRNA (channel I, Vout 5 0.89 mL/min, Vc 5 5.9 mL/min, andt0 5 0.21 min).were analyzed by FFF. The three last eluted peaks in

NILSSON, BULOW, AND WAHLUND: FLOW FFF IN QUANTITATION OF RIBOSOME AND SUBUNITS 463

completely dissociated into subunits (Spirin et al., 1970).Hence, only the two subunit peaks at 17 and 20 nm wereobserved. Further confirmation of the identity of the twosubunit peaks in fractogram 1 was obtained by analyzingpurified subunits isolated by ultracentrifugation (Fig. 2,fractograms 3 and 4). The small and large subunits gavehydrodynamic diameters of 18 and 20 nm, respectively.

Direct evidence for the identity of the ribosomalpeaks was obtained by electron microscopy of the col-lected fractions corresponding to the small subunit, thelarge subunit, and the ribosome. Average diameters of17.1 6 0.6 (n 5 2 3 42), 19.8 6 0.6 (n 5 2 3 53), and21.1 6 0.8 nm (n 5 2 3 34) were obtained.

Nonribosomal Peaks

Figure 3. Growth phases. The insert shows the growth curve con-The peak at 5 nm (Fig. 2, fractogram 1) represents thestructed from 10 measurements where data points were connectedtRNA fraction (molecular weight about 25,000) of thewith straight lines to indicate the trend. Samples were withdrawn at

cell as supported by separate analysis of a standard times indicated by arrows whose numbers indicate the fractogramsample of E. coli tRNA (Fig. 2, fractogram 5). The obtained. Channel IV was used for all fractograms and the flow rates

and void times were: fractogram 1—Vout 5 0.56 mL/min, Vc 5 5.3ultraviolet (UV) spectra of the 5-nm peak in the stan-mL/min, and t0 5 0.22 min; fractogram 2—Vout 5 0.54 mL/min,dard and in the E. coli sample both displayed a typicalVc 5 5.3 mL/min, and t0 5 0.22 min; fractogram 3—Vout 5 0.54 mL/nucleic acid maxima at 258 nm.min, Vc 5 5.3 mL/min, and t0 5 0.22 min; fractogram 4—Vout 5 0.53

The peak at approximately 10 nm (Fig. 2, fractogram mL/min, Vc 5 5.3 mL/min, and t0 5 0.22 min.2) contains proteins released in the preparation proce-dure. This is supported by the UV spectrum of thepeak showing a maximum at 280 nm. Sodium dodecyl

We found significant differences in the number ofsulfate–polyacrylamide gel electrophoresis (SDS–ribosomes per cell between all phases except betweenPAGE) of a collected fraction also showed the presencethe stationary and the log phase (Table I). This confirmsof several protein bands.earlier observations that the number of ribosomes percell changes during the cell cycle (Mendelsohn and Tis-sieres, 1959). This parameter has been determined byRepeatabilityother methods and is known to increase in correlation

The analysis of the sample of a preparation made in to the growth rate in the logarithmic phase (Bremer10 mM Mg21 was repeated 10 times using the same appa- and Dennis, 1987; Liveris et al., 1991). At the growthratus. The mass fraction of ribosome was determined rate corresponding closest to that in Table I (one dou-with a relative standard deviation (srel%) of 4.6%. The bling per hour), the number of ribosomes per cell pre-number of ribosomes per cell was determined with an viously reported was 4777 (Bremer and Dennis, 1987)srel% of 9.6%. These results indicate that changes in the and 25,000 (Liveris et al., 1991), which shows a fairmass fraction of ribosome larger than about 5% may be agreement with our data. However, since differentjudged significant. Likewise, changes in the number of strains and growth media were used, the results are notribosomes larger than 10% can be detected. The hydro- directly comparable. Furthermore, in the log phase thedynamic diameter was determined with an srel% of 5.4%. peak at 38 nm may represent polysomes, indicating the

presence of more polysomes in this phase than in theother phases.Growth Phases

The differences in mass fraction of ribosome observedin the various growth phases were all significant (seeSamples were withdrawn from an E. coli culture in the

lag phase, the logarithmic (log) phase, the stationary Table I). The highest values were observed in the logand stationary phases, where the cells are most activephase, and the declining phase (Fig. 3, insert). Ribo-

somes were prepared, followed by FFF analysis. In all (high protein production capacity), and the subunitsthus should be associated to ribosomes to a higher de-growth phases the three peaks characteristic of the ribo-

somal species were detected (Fig. 3). Responses for the gree than in the lag phase. Further, in the declinationphase (low protein synthesis) the ribosomes must haveribosomal species varied significantly between the vari-

ous growth phases. This reflects both the differences in been more dissociated to free subunits than in the logand stationary phase. This behavior is expected since itthe number of cells per volume of the culture and in

the number of ribosomes per cell. has been shown that a low growth rate gives a lower

464 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 54, NO. 5, JUNE 5, 1997

Table I. Mass fraction of ribosome and the number of ribosomes in E. coli TG1 at differentphases of growth.a

dH for the 30S, 50S, and Mass fraction of Number of ribosomesPhase 70S particles (nm)b ribosome per cell

Lag phase 17; 19; 23 0.43 (0.032) 195 (45)Logarithmic phase 18; 20, 25 0.72 (0.043) 4100 (1400)Stationary phase 18; 20; 25 0.81 (0.023) 3500 (1400)Declination phase 17; 20; 24 0.37 (0.038) 25 (15)

aReported values are averages of six analyses, three from each of two parallel preparations,except for the lag phase experiment, which is based on six analyses of one preparation. Thestandard deviation is given within parentheses.

bdH is the hydrodynamic diameter.

mass fraction of ribosome than a high growth rate (Birn- added to the culture and after 4.1 h of further cultivationribosomes were prepared and analyzed by FFF (Fig. 4,baum and Bailey, 1991; Jinks-Robertson and Nomura,

1987; Maaløe, 1979). fractogram 2).The fractograms also give valuable information about The only slightly higher OD550 obtained after the ad-

the internal tRNA levels, which is useful in evaluating dition of the antibiotic, as compared to before the addi-the protein-synthesizing status of the cell. The response tion, shows that the cell growth had ceased. This meansof the 5-nm peak is hence very small in the lag phase that protein synthesis should have stopped, which(Fig. 3, fractogram 1). This may in part be due to the should lead to less ribosomal protein and hence fewerlow number of cells in this growth phase, but when ribosomes. We did observe the expected decrease in thenormalized to the number of cells, it was found to be number of ribosomes per cell after the addition of themuch lower (about 10-fold) than in the other growth antibiotic (Table II). However, some ribosomes still re-phases. This indicates that the tRNA concentration in mained intact (70S). This is likely due to the fact thatthe lag phase is lower than in the other growth phases. although inactive in protein synthesis, the ribosomes

have not yet dissociated into free subunits.Antibiotic-Treated Cells The mass fraction of ribosome increased after the

addition of the antibiotic. However, this is only causedChloramphenicol is known to inhibit peptide bond for-by the selective decrease of the mass fraction of themation in bacteria by binding to the large ribosomallarge subunit, as demonstrated in Figure 4, because itsubunit (Vasquez, 1964). We investigated the effect ofis the target for chloramphenicol. Fractogram 2 in Figurechloramphenicol on the ribosomal composition of E.4 shows that the peak height of the large subunit de-coli by analyzing a sample from a culture in the logcreased remarkably relative to the small subunit andphase (Fig. 4, fractogram 1). Then chloramphenicol wasthe ribosome but the relative amounts of the small sub-unit and the ribosome were unchanged. Therefore theobserved increase in mass fraction of ribosome is notreflecting an increased ribosomal activity but merely thedecreased amount of large subunits, and this means thatthe mass fraction of ribosome in this case is not a rele-vant parameter for evaluating the protein-synthesizingactivity.

Table II. Mass fraction of ribosome and the number of ribosomesin E. coli TG1 before and after addition of chloramphenicol.a

Mass fraction Number of ribosomesSample of ribosome per cell

Figure 4. Antibiotic-treated cells. Fractogram 1: before addition of Before addition of 0.70 (0.0061) 10,300 (500)chloramphenicol (channel III, Vout 5 0.54 mL/min, Vc 5 5.3 mL/min, chloramphenicoland t0 5 0.22 min). The sample was taken at 5.5 h in the growth cycle. After addition of 0.79 (0.004) 5,100 (900)Fractogram 2: after addition of chloramphenicol (channel III, Vout 5 chloramphenicol0.60 mL/min, Vc 5 5.3 mL/min, and t0 5 0.22 min). The sample wastaken at 9.6 h in the growth cycle. Chloramphenicol had been added aValues reported are averages of triplicate analysis of one sample

of bacteria. The standard deviation is given within parentheses.at 5.5 h in the growth cycle.

NILSSON, BULOW, AND WAHLUND: FLOW FFF IN QUANTITATION OF RIBOSOME AND SUBUNITS 465

Table III. Mass fraction of ribosome and the number of ribosomes mids as control (Fig. 5, fractogram 2). The number ofin E. coli TG1 at various genetical modifications.a ribosomes per cell increased when the plasmid was in-

duced (see Table III). This increase can be due to theMass fraction Number of ribosomesSample of ribosome per cell increased demand for ribosomes to synthesize the pro-

tein encoded by the plasmid. The mass fraction of ribo-Plasmid-free strain 0.92 (0.01) 20,100 (600)

some decreased for the bacteria carrying induced plas-Noninduced plasmid p1 10,100 (2200)mids as compared to the control. The number of freeInduced plasmid 0.69 (0.02) 15,300 (1500)subunits has thus increased after induction of the plas-

aValues reported are averages of two analysis of one sample of mids. This may be due to the short mRNA moleculesbacteria. The standard deviation is given within parentheses.

generated from the plasmids. After completing transla-tion, the ribosomes leave the mRNA and appear in theform of free subunits.

Figure 5 also demonstrates that the FFF separationsE. coli-Harboring Protein-Encoding Plasmids were finished in less than 14 min, a much shorter analysis

time than in the established method of ultracentrifuga-In genetic engineering work plasmids are frequently tion. The field-flow fractionation method therefore of-used for bacterial protein production. In this case the fers a rapid and attractive alternative for controllinglevel of expression is controlled by inducing a promoter protein production in recombinant bacteria.upstream of the structural gene. To optimize such pro-duction, an understanding of the protein-synthesizingmachinery will be essential. The effect of introducing

We are grateful to Dr. Eric Carlemalm at the Electron Mi-plasmids was examined by monitoring the ribosomalcroscopy Unit, Lund University for operating the micro-composition and the number of ribosomes per cell ofscope.E. coli TG1 harboring noninduced plasmids and of the

plasmid-free strain. The results in Table III show thatthe number of ribosomes per cell decrease significantlyin the presence of plasmids which can be explained by

Referencesthe burden on cell metabolism caused by the plasmids.The plausible effect is that more energy is directed Birnbaum, S., Bailey, J. E. 1991. Plasmid presence changes the relative

levels of many host cell proteins and ribosome components in re-toward maintaining the plasmid DNA than towardcombinant Escherichia coli. Biotechnol. Bioeng. 37: 736–745.protein synthesis and thus less ribosomes are synthe-

Bremer, H., Dennis, P. P. 1987. Modulation of chemical compositionsized.and other parameters of the cell by growth rate, pp. 1527–1542. In:

Furthermore, we monitored the ribosomal composi- F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik,tion of bacteria having induced plasmids (Fig. 5, frac- M. Schaechter, and H. E. Umbarger (eds.), Escherichia coli and

Salmonella typhimurium. ASM, Washington, DC.togram 1) and used the bacteria with noninduced plas-Carlsson, H., Prachayasittikul, V., Bulow, L. 1993. Zinc ions bound

to chimeric His4/lactate dehydrogenase facilitate decarboxylationof oxaloacetate. Prot. Eng. 6: 907–911.

Giddings, J. C. 1993. Field-flow fractionation: Analysis of macromo-lecular, colloidal, and particulate materials. Science 260: 1456–1465.

Hauge, J. G. 1971. Pressure-induced dissociation of ribosomes duringultracentrifugation. FEBS Lett. 17: 168–172.

Jinks-Robertson, S., Nomura, M. 1987. Ribosomes and tRNA,pp. 1358–1385. In: F. C. Neidhardt, J. L. Ingraham, K. B. Low, B.Magasanik, M. Schaechter, and H. E. Umbarger (eds.), Escherichiacoli and Salmonella typhimurium. ASM, Washington, DC.

Litzen, A. 1993. Separation speed, retention, and dispersion in asym-metrical flow field-flow fractionation as functions of channel dimen-sions and flow rates. Anal. Chem. 65: 461–470.

Liveris, D., Klotsky, R.-A., Schwartz, I. 1991. Growth rate regulationof translation initiation factor IF3 biosynthesis in Escherichia coli.J. Bact. 173: 3888–3893.

Maaløe, O. 1979. Regulation of the protein-synthesizing machinery—ribosomes, tRNA, factors, and so on, pp. 487–542. In: R. F. Gol-berger (ed.), Biological regulation and development, vol. 1. Plenum,New York.

Mendelsohn, J., Tissieres, A. 1959. Variations in the amount of ribo-Figure 5. Cells harboring protein-encoding plasmids. Fractogram 1:preparation of bacteria with induced plasmid (channel V, Vout 5 nucleoprotein particles in Escherichia coli. Biochim. Biophys. Acta

35: 248–250.0.88 mL/min, Vc 5 5.1 mL/min, and t0 5 0.23 min). Fractogram 2:preparation of bacteria with noninduced plasmid (channel V, Vout 5 Nilsson, M., Birnbaum, S., Wahlund, K.-G. 1996. Determination of

relative amounts of ribosome and subunits in Escherichia coli using0.88 mL/min, Vc 5 5.1 mL/min, and t0 5 0.23 min).

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asymmetrical flow field-flow fractionation. J. Biochem. Biophys. monoribosomes of Escherichia coli in the presence of a reducedmedium magnesium ion concentration. Mol. Biol. 4: 501–508.Methods 33: 9–23.

Pettersson, I., Hardy, S. J. S., Liljas, A. 1976. The ribosomal protein Vasquez, D. 1964. The binding of chloramphenicol by ribosomes fromBacillus megaterium. Biochem. Biophys. Res. Commun. 15: 464–L8 is a complex of L7/L12 and L10. FEBS Lett. 64: 135–138.

Spirin, A. S. 1971. On the equilibrium of the association-dissociation 468.Wittgren, B., Wahlund, K.-G., Derand, H., Wesslen, B. 1996. Aggrega-reaction of ribosomal subparticles and on the existence of the so-

called ‘‘60 S intermediate’’ (‘‘swollen 70 S’’) during centrifugation tion behavior of an amphiphilic graft copolymer in aqueous mediumstudied by asymmetrical flow field-flow fractionation. Macromole-of the equilibrium mixture. FEBS Lett. 14: 349–353.

Spirin, A. S., Sofronova, M. Yu., Sabo, B. 1970. Dissociation of 70 S cules 29: 268–276.

NILSSON, BULOW, AND WAHLUND: FLOW FFF IN QUANTITATION OF RIBOSOME AND SUBUNITS 467