Biotechnology Letters 23: 839–843, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Real time detection and quantification of inclusion bodies expressed inEscherichia coli by impedance measurements

Pramod Upadhyay∗, Ashok K. Patra, Rituparna Mukhopadhyay & Amulya K. PandaNational Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India∗Author for correspondence (Fax: + 91-11-6162125; E-mail: pkumar@nii.res.in)

Received 30 January 2001; Revisions requested 27 February 2001; Revisions received 19 March 2001; Accepted 22 March 2001

Key words: E. coli, high cell density, human growth hormone, impedance, inclusion bodies, real time estimation

Abstract

Escherichia coli expressing human growth hormone as inclusion bodies was cultured in a fermenter. Real time,non-invasive detection and quantification of inclusion body protein expressed in E. coli was performed by im-pedance measurements at 50 MHz and 180 MHz. At 50 MHz rotation of dipoles of the protein and their protonfluctuation, i.e., β-dispersion of protein aggregates formed inside the cell as a result of expressed protein, resultsin an additional decrease in impedance. At 180 MHz the impedance remained at a plateau. In a high cell densityE. coli culture, after induction with IPTG, when the cell mass remained unchanged, an increase in the magnitude ofβ-dispersion was observed at 50 MHz. This was due to the formation and subsequent increase in the concentrationof r-human growth hormone which aggregate as inclusion bodies. The estimation of inclusion bodies by taking theratio of impedance at 180 MHz and at 50 Mhz matched with the amount of protein estimated after extraction andpurification (coefficient of correlation was 0.92). This is the first report of real time detection and monitoring ofrecombinant protein expressed as inclusion bodies by impedance measurements.

Introduction

Escherichia coli is widely used for the productionof recombinant proteins. Usually a high level of ex-pressed recombinant proteins accumulate as insolubleaggregates inside the cell in the form of inclusion bod-ies. In general, the protein concentration in inclusionbodies is estimated by harvesting the cells followed byisolation and purification of inclusion bodies (Mart-son 1986). This procedure of protein estimation iscumbersome and time consuming. It is of increasingconcern to develop methods for the detection as wellas quantification of inclusion body proteins inside thecell during fermentation. Light scattering has beenused for such an application (Wittrup et al. 1988).There are a number of recent reports for the real timequantification of protein production in which usinggreen fluorescent protein (GFP) (Albano et al. 1998,Daabrowski et al. 1999, DeLisa et al. 1999), elec-trochemical sensor (Biran et al. 1999) and infraredspectroscopy (Crowley et al. 2000) have been used.

Impedance methods for the measurement of to-tal biomass in a reactor have been known for a longtime (Kell & Davey 1990). This paper describes anapplication of impedance measurements to detect andquantify recombinant proteins expressed as inclusionbodies in E. coli. Each protein molecule possessesa permanent dipole moment which will experiencean orientational force when subjected to an alternat-ing electric field (Grant et al. 1978). The constantBrownian motion of the molecules will randomizethis alignment and the magnitude of the additionalpolarization will depend on the extent of these two ef-fects. This polarization is also known as β-dispersion.The magnitude of β-dispersion is proportional to theconcentration of the protein. Expressed recombinantproteins have a unique β-dispersion due to the ro-tation of dipoles of the protein (Grant et al. 1978).This dispersion was observed by recording the im-pedance from 1 MHz to 180 MHz for recombinanthuman growth hormone expressed as inclusion bodiesin E. coli during high cell density fermentation. In a

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Fig. 1. Schematic diagram of various interconnections of fermenter,bubble trap, peristaltic pumps, impedance analyser and computerinterface for the on-line estimation of r-hGH expressed as inclusionbodies.

sample containing an additional unique protein (inclu-sion bodies) an excessive lowering of impedance wasobserved between 20–100 MHz, which was due to theβ-dispersion of inclusion body proteins. At frequen-cies higher than 100 MHz, changes in impedance weredue to changes in total biomass. The β-dispersion maybe visualized as impedance connected in parallel to theimpedance of the rest of the cell mass and medium. Inthis paper the impedance was read both at 50 MHzand 180 MHz. At 50 MHz the impedance (Z) couldbe approximated as

1/Z50 MHz = 1/Z50 MHz (cell mass and medium) +1/Z(50 MHz) inclusion bodies. (1)

At 180 MHz the excess contribution due to inclusionbodies is negligible,

Z(180 MHz) = Z(180 MHz) (cell mass and medium). (2)

It can be seen from Equations (1) and (2) thatthe ratio of Z180 MHz and Z50 MHz is proportional tothe impedance contributed by inclusion bodies. Inorder to simplify the methodology, inclusion bod-ies were detected and quantitated in real time, whencell mass remained unchanged. In such a situationZ180 MHz/Z50 MHz is a direct measure of amount ofinclusion bodies. The correlation coefficient was 0.92when Z180 MHz/Z50 MHz and the amount of proteinestimated after extraction and purification were com-pared. This method gives the average concentration

Fig. 2. Impedance-frequency plot of two E. coli samples havingidential cell mass, before induction (�) and 3 h after induction (◦).Each data point is an average of 10 measurements and error barsshow the standard deviation.

of inclusion body protein. It is similar to the methodin which GFP is used as a reporter without a fluo-rescent tag on the expressed protein. This method isnon-intrusive and allows real time monitoring of theprotein expression as inclusion bodies.

Materials and methods

Fermentation

Recombinant E. coli (Mukhija et al. 1995) was grownin a 3.5 l fermenter (2 l working volume) in complexmedium (Mori et al. 1979), except that glucose andyeast extract were each at 10 g l−1. Fermentation wascarried out at 37 ◦C with vigorous aeration and themedium was maintained at pH 7 throughout the fer-mentation. After 3 h batch growth, continuous supplyof glucose and yeast extract was commenced to main-tain the specific growth rate of cells at 0.2 h−1. Thedetails of fed-batch fermentation strategy is describedelsewhere (Patra et al. 2000). After 6 h the turbiditymeasured at 600 nm was 40 (dry cell wt 16 mg ml−1),the culture were induced with 1 mM isopropyl thio-galacto pyranoside (IPTG) and cultivated for another4 h. The harvested cells were checked for expressionand processed for the purification of inclusion bodies.

Off-line impedance measurement

Off-line impedance measurements were performedon E4916A impedance/LCR meter (Hewlett-Packard).

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Fig. 3. Real time monitoring of high cell density E. coli fermentation expressing r-hGH. (A) Shows a comparison of dry cell weight (◦) and Z(•) at 50 MHz and in (B) dry cell weight (◦) versus Z (•) at 180 MHz is shown. In (A) and (B) time of induction with IPTG was 6 h.

E. coli cells expressing recombinant human growthhormone (r-hGH) were used. The impedance of theculture was measured in the frequency range 1 to180 MHz. Prior to the measurement, samples werediluted to equalize their turbidity and average of 10measurements was read.

On-line impedance measurement

The schematic diagram of various interconnections isshown in Figure 1. Pumps were stopped during themeasurement and flushed after the measurement wasover. One measurement cycle took 20–30 s and duringthis time the temperature change was less than 0.5 ◦C.

SDS-PAGE and protein estimation

The inclusion bodies were processed by proceduresreported earlier (Khan et al. 1998). Induced E. colicells sampled at different time points during fed-batchfermentation were centrifuged at 4000 g for 30 minand the cell pellet was suspended in 50 mM Tris-HCl buffer (pH 8) containing 5 mM EDTA and 1 mMPMSF. Cells were lysed by sonication and centrifugedat 8000 g for 30 min to isolate r-hGH inclusion bod-ies. The inclusion bodies were completely soluble in50 mM Tris/HCl buffer (pH 8.5) containing 1% SDS.

The concentration of protein in inclusion bodies wasestimated by bicinchonimic acid method as describedpreviously (Smith et al. 1985) and SDS-PAGE wascarried out (Laemmli 1970).

Results and discussion

The expression of r-human growth hormone after in-duction with IPTG was confirmed by SDS-PAGE. Toidentify the optimal set of frequencies for monitor-ing β-dispersion due to inclusion bodies, impedancewas measured at different frequencies. Two samples,one without inclusion bodies (before induction) andthe other with inclusion bodies (after induction), wereused. Equal amounts of E. coli cells were present inthese diluted samples. It was confirmed by the drycell weight of the cell mass (results not shown). Fig-ure 2 shows an impedance (Z)-frequency plot of twoE. coli samples, one before induction (�) and theother after 3 h of induction (©). Both the samplesshow similar absorbance at frequencies lower than5 MHz and higher than 100 MHz. However, between5 MHz and 100 MHz they absorb differently, due tothe presence of inclusion bodies in the post inductionsample. A comparison of impedance of these two sam-

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Fig. 4. A comparison of amount of r-hGH (•) expressed as inclusion bodies estimated after extraction, purification and Z180 MHz/Z50 MHz (◦)during high cell density cultivation of E. coli. In the inset SDS-PAGE analysis is shown. Lanes 1 and 2 show uninduced cells. Lanes 3–6 showinduced cells after 1, 1.5, 3 and 4 h of IPTG induction respectively and in lane 7, MW markers in kDa is shown. The time of induction withIPTG was 6 h.

ples confirmed that the presence of inclusion bodiescan be identified by impedance measurements in thefrequency range 5 to 100 MHz.

A selective increase in the absorption of electri-cal energy around 50 MHz, as seen by the decreasein Z was due to the formation of protein whichwas not present initially. The rotation of dipoles ofnewly formed protein lead to an ‘extra absorption’at 50 MHz making its detection and quantificationpossible. For this newly formed protein to cause anincrease in turbidity, its size has to be at least of theorder of the wavelength of light used to measure theturbidity. There is, however, no such constraint whenimpedance is recorded. As soon as protein dipoles areformed, they absorb 50 MHz electrical signal leadingto β-dispersion and their detection and quantificationbecomes possible much before they cause a change inturbidity.

Subsequent measurements of impedance were car-ried out at 50 MHz and 180 MHz and entire growthwas monitored by impedance and turbidity. Figure 3

shows growth kinetics of E. coli expressing r-hGHas inclusion bodies. In Figure 3A, a comparison ofdry cell weight and Z at 50 MHz is shown and inFigure 3B, dry cell weight verses Z at 180 MHz isshown. At 180 MHz, Z values follow dry cell wt,indicating that the total biomass can be monitored.At 50 MHz, Z values start decreasing after the in-duction of cells with IPTG. This decrease was dueto the β-dispersion of newly expressed protein whichaccumulates as inclusion bodies.

The plateauing of cell mass after induction as con-firmed by turbidity, dry cell weight and Z enabled usto look for a simple relationship between the Z val-ues after induction and the concentration of inclusionbody protein. A plot of Z180 MHz/Z50 MHz and timeis shown in Figure 4. In this plot there is a changein the slope at 6.75 h, that is 45 min after induc-tion. This change of slope, which was due to theexpression of the protein, is a signature of extra β-dispersion. The magnitude of Z180 MHz/Z50 MHz afterthe inflection point correlate with the amount of pro-

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tein estimated by BCA protein assay, also plotted onthe same graph. From the plot of amount of proteinand Z180 MHz/Z50 MHz (not shown), the amount ofprotein (g l−1) = 15.2–13.8(Z180 MHz/Z50 MHz) andthe coefficient of correlation was 0.92.

This technique of monitoring expressed proteinby measuring impedance at two frequencies can beextended to a number of proteins which give β-dispersion or absorb in the frequency range 10–100 MHz. The technique will find applications inmolecular biology laboratories and also in industriesmaking products based on recombinant proteins.

Acknowledgements

We thank Mr Sosil Tyagi and Hewlett-Packard In-dia Limited for providing us E4916A for impedancemeasurements. Dr Lalit Garg of National Instituteof Immunology provided us the recombinant E. coliexpressing human growth hormone. This work wassupported by the core grant of Department of Biotech-nology, India, to the National Institute of Immunology.

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