Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli.

The large mechanosensitive ion channel (MscL) of Escherichia coli was expressed on a plasmid encoding MscL as a fusion protein with glutathione S-transferase in an Escherichia coli strain containing a disruption in the chromosomal mscL gene. After purification of the fusion protein using glutathione-coated beads, thrombin cleavage allowed recovery of the MscL protein. The purified protein was reconstituted into artificial liposomes and found to be fully functional when examined with the patch-clamp technique. The reconstituted recombinant MscL protein formed ion channels that exhibited characteristic conductance and pressure sensitivity and were blocked by the mechanosensitive ion channel inhibitor gadolinium. The recombinant MscL protein was also used to raise specific anti-MscL polyclonal antibodies which abolished channel activity when preincubated with the MscL protein.


Purification and Functional Reconstitution of the Recombinant Large Mechanosensitive Ion Channel (MscL) of Escherichia coli*
(Received for publication, March 29, 1995, and in revised form, May 22, 1995) Claudia C. Haser, Alexander C. Le Dain, and Boris Martinac § The large mechanosensitive ion channel (MscL) of Escherichia coli was expressed on a plasmid encoding MscL as a fusion protein with glutathione S-transferase in an Escherichia coli strain containing a disruption in the chromosomal mscL gene. After purification of the fusion protein using glutathione-coated beads, thrombin cleavage allowed recovery of the MscL protein. The purified protein was reconstituted into artificial Iiposomes and found to be fully functional when examined with the patch-clamp technique. The reconstituted recombinant MscL protein formed ion channels that exhibited characteristic conductance and pressure sensitivity and were blocked by the mechanosensitive ion channel inhibitor gadolinium. The recombinant MscL protein was also used to raise specific anti-MscL polyclonal antibodies which abolished channel activity when preincubated with the MscL protein.
Mechanosensitive ion channels have been found in organisms of different phylogenetic origin including animals, plants, fungi, and bacteria (1)(2)(3)(4), Although exclusively documented in patch-clamp experiments, the ubiquity of mechanosensitive channels suggests that they have important physiological functions in various types of biological cells, Increasing evidence indicates that the physiological role of these channels is to modulate cell responses to mechanical stimuli such as stretch, contraction, or osmotic stress (5)(6)(7), Microorganisms, such as the enterobacterium Escherichia coli, are constantly exposed to changes in environmental osmolarity, A recent study by Berrier et at. (7) demonstrated that the loss of metabolites following osmotic down-shock was blocked by gadolinium, the well documented inhibitor of mechanosensitive channels (4), Patch-clamp studies of giant spheroplasts of E. coli have revealed the presence of two distinct types ofmechanosensitive ion channels in the bacterial cell envelope: a small weakly anion-selective mechanosensitive channel (MSCS)1 with a conductance of approximately 1 nS (8,9) and a large nonselective channel (Mscl.) with a conductance of 2,5-3.0 nS (9). Both ion channels could be reconstituted into artificialliposomes either by fusing bacterial membrane vesicles (9,10) or by reassembly of detergent-solubilized membrane extracts (9,11), without * This work was supported in part by Australian Research Council Grant A19332733. The costs of publication of this article were defrayed in part by the payment of page charges, This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In the present study, we have used a common method for expressing recombinant proteins in E. coli to produce significant amounts of purified MscL protein (14,15). The recombinant protein was found to be fully functional when reconstituted into artificialliposomes and was used to raise polyclonal anti-MscL antibodies. This work was presented in preliminary form (16),
DNA Preparation, Manipulation, and Analysis-Plasmid DNA was extracted from E. coli cells using the alkaline lysis method (17). Standard techniques (18) were used for the generation of recombinant plasmid constructs described under "Results." Restriction enzymes and DNA ligase were purchased from Promega and used as specified by the manufacturer. DNA restriction fragments were electrophoresed in horizontal 0.8% agarose gels in 40 mM Tris, 1 mM EDTA, pH 8.0, and stained with ethidium bromide (0.5 ug/rnll, DNA fragments were excised from 1% low melting agarose gels (Promega), melted at 55 "C, and used directly in ligation reactions.
Protein Purification and Analysis-Recombinant fusion protein was purified essentially as described previously (14,15). Bacterial cells harboring the plasmid pGEX1.1 were subgrown for 1 h at 37°C (1 ml of an overnight culture in 20 ml of broth), and fusion protein gene expression was induced for 3 h with 0.1 mM IPTG. The cells were harvested, resuspended in 5 ml of 150 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 8.0, and lysed by addition oflysozyme (0.1 mg/ml) and detergent (1.5% octyl glucoside). After bath sonication for approximately 60 s (Unisonics Pty. Ltd., Sydney, Australia), cell debris was pelleted (16,000 rpm, 20 min; 18329 This is an Open Access article under the CC BY license. J2-MI, Beckman), and 0.5 ml of glutathione-Sepharose 4B beads were added to the supernatant for 1 h at room temperature (20-22°C). The beads were then washed at least three times (by centrifugation using a desk top centrifuge at 4,000 rpm for 5 min) in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2HPO., 1.4 mM KH 2PO., pH 7.2, adjusted with NaOH) and resuspended in PBS containing an additional 150 mM NaCl, 2.5 mM CaCI 2 , and 50 mM Tris. Thrombin was added to a final concentration of = 1 unit!/Lgof protein and incubated for 1 h at room temperature. Octyl glucoside in PBS was added to a final concentration of 1% (w/v) for 10 min, the beads were pelleted (4,000 rpm, 5 min), and the supernatant was found to contain the MscL protein. For reconstitution into artificial liposomes and antibody generation, purified MscL protein was dialyzed for 16 h against 4 liters of 10 mM Tris, 1 mM EDTA, pH 7.5, with Calbiosorb beads to remove any octyl glucoside, and the protein was concentrated in Amicon filtration units (Amicon, Inc.), Protein concentrations were determined using the Dc protein assay (Bio-Rad), and protein samples were analyzed by 12% SDS-polyacrylamide gel electrophoresis as described (18). The N-terminal amino acid sequence of the purified recombinant MscL protein was analyzed at the University of Western Australia Centre for Molecular Biology, using the Edman degradation procedure.
Reconstitution of Recombinant Proteins in Artificial Liposomes-Liposomes were prepared using a method similar to that described previously (9,10). Briefly, phosphatidylcholine with 10% cholesterol was dissolved in chloroform. Small aliquots of lipid were dried under nitrogen, resuspended in 5 mM Tris, pH 7.2, and bath-sonicated for 15 min. The liposomes were collected by ultracentrifugation (TL-100, Beckman Instruments) at 105,000 x g for 1 h and resuspended in 10 mM MOPS, 5% ethylene glycol, pH 7.2. Purified protein was added at the desired protein:lipid ratio. Aliquots of the liposomes were spotted onto glass slides and allowed to dehydrate for several hours followed by overnight rehydration (200 mM KCl, 5 mM HEPES, pH 7.2) under humid conditions.
Production and Purification ofMscL-specific Polyclonal Antibodies-Two female New Zealand albino rabbits were prebled and injected with approximately 100 p.g of purified MscL protein in TiterMax adjuvant (Vaxcel, Inc., Norcross, GA). Antibody titers were checked after 3 and 6 weeks by Western blot analyses essentially as described (18). Briefly, protein samples were electrophoresed on 12% SDS-polyacrylamide gels and transferred to nitrocellulose in transfer buffer (20 mM Tris, 150 mM glycine, 20% methanol). The filters were reacted with rabbit antibodies (diluted 1:1100 for whole sera, 1:50 for affinity-purified antibodies), incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Sigma) (diluted 1:1,000), and developed with substrate solution (0.2 mM o-dianisidine, 0.01% H 2 0 2 in 10 mM Tris, pH 7.4). MscL-specific antibodies were purified by coupling purified MscL protein to cyanogen bromide-activated Sepharose beads with subsequent washes and elution conditions as described previously (19). Affinity-purified antibodies were dialyzed against 100-fold diluted PBS and then concentrated 100-fold by lyophilization (Dynavac, Pty. Ltd., Sydney, Australia). For enzyme-linked immunosorbent assay (ELISA), the wells of microtiter plates were coated with various concentrations of purified MscL protein in PBS overnight at 4°C. The coated plates were then washed in PBS with 0.02% Tween, blocked with 2% bovine serum albumin in PBS for 2 h at room temperature, washed again, and reacted with antisera (diluted 1:1,000 for whole sera, 1:100 for affinity-purified antibodies) at 4°C overnight. The plates were washed again and incubated with peroxidase-labeled secondary antibody (diluted 1:5000) for 2 h at room temperature. Substrate was added, and absorbance at 410 nm was measured on a microplate reader (Series 700, Cambridge Technology, Inc., Watertown, MA).
Electrophysiological Recordings-The improved patch-clamp techniques of Hamill et al. (20) were used to record single-channel currents from isolated membrane patches. Pipettes were made from borosilicate glass (Drummond Scientific Co., Broomall, PAl using a FlaminglBrown Micropipette puller (P-87, Sutter Instrument Co., Novato, CAl and pulled to a diameter which gave bubble numbers of 3.2-3.5 in 100% ethanol, corresponding to pipette resistances in the range 6. Wetzlar, Germany) and were touched against unilamellar blisters arising spontaneously from the liposomes as reported (9,10). Seals (>20 GO) either formed immediately or following application of a brief (1-2 s) pulse of negative pressure «50 mmHg), applied to the interior of the patch pipette.
Channel activation was normally achieved by applying pressure steps of -20 to -30 mmHg by mouth over a period of several seconds every 5-10 s. For rapid pressure steps (=1 s), suction was applied by mouth in a single step from 0 mmHg to a pressure exceeding the activation threshold for that particular patch.
Single-channel currents were filtered at 1 kHz, recorded using a patch-clamp amplifier (List Electronics, Darmstadt, Germany), and digitized at 5 kHz by a computer running WinTida analogue to digital acquisition software (Heka Electronics, Heidelberg, Germany). Data files were analyzed off-line using commercial software or programs written in this laboratory.

Construction of a Plasmid Encoding a GST-MscL Fusion
Protein-The XhoI DNA restriction fragment containing the entire ORF of mscL from plasmid p5-2-2 (12) was first subcloned into plasmid vector pGEMllZf( +) in the desired orientation as determined by restriction enzyme analysis. The gene was then excised and cloned into the expression vector pGEX-2T as a BamHI-EcoRI DNA fragment (now named pGEX1.1), thus generating a continuous ORF with the GST gene (Fig. 1). This genetic construct should lead to the production of a 41-kDa hybrid protein consisting of aN-terminal GST portion (26 kDa) and a C-terminal MscL portion (15 kDa) separated by a thrombin cleavage site. Upon thrombin cleavage of the fusion protein, nine amino acids as well as the initial methionine residue, which are not present in purified wild-type MscL (12), are expected to be present on the N terminus of the recombinant MscL protein (Fig. 1). N-terminal protein sequence analysis of the purified MscL protein confirmed the presence of these ten amino acid residues.
Purification of the GST-MscL Fusion Protein-Upon IPTG induction of E. coli cells carrying the plasmid pGEX1.1, encoding the GST-MscL fusion protein, a major protein band of approximately 40 kDa appeared in SDS-polyacrylamide gel electrophoresis analysis (Fig. 2A, lane 2). The induced fusion protein was purified in a single step by addition of glutathione-FI G. 2. A , SDS-polyacrylamide gel electrophoresis protein patterns during purification steps ofthe GST-MscL fusion protein from E. coli. B , West ern blot using immunoaffinity-purified polyclonal anti-MscL antibodies. Lan es: 1, total E. coli cells before induction with IPTG; 2, total E. coli cells after induction with IPTG; 3, gluta thione-Sepharose beads absorbed material; 4 , thr ombin cleavage of glutathione-Sepharose beads absorbed material; 5   Pressure S ensitivity of th e MscL-Ch annels were activated wh en negative pr essure (suction ) excee ded a threshold, typically in th e range of 50 to 100 mmHg. As th e amount of applie d pr essure increased, channel activity and hence channel open prob ability a lso increased (F ig. 4A ). We hav e us ed a Boltzmann distribution curve to describe th e a ppa re nt pr essure se ns itivity of th e reconsti tuted MscL protein . Th e open probability of th e ch annels in a particul ar patch was plotted agains t th e a pplied suct ion, a nd th e dat a wer e fitted to a Boltzmann distribution (Fig. 48). For recon stituted MscL, an e-fold change in open pr obability wa s obse rve d following a chan ge of 4.9 :!: 1.4 mmHg (rn ean :t S .E ., n = 3 patches) a t a pipette pot ential of + 10 mY, a nd 3.9 :!: 1.1 mmHg at a pip ette pot en tial of -10 mV (n = 2 patches). Th e ave rage a pplied negative pr essure required to induce half-maximal activation ofMscL wa s 72 :!: 3 mmHg (n = 3 patches ) a t a pip ette pot ential of + 10 mV, and 71 :!: 6 mmHg (n = 2 patches ) at a pip ette pot en tial of -10 mV.
In response to a mor e r apid (1-2 s ) change in pressure, th e MscL exh ibite d rapid activation , followed by ada pta t ion (Fig.  5). At present, we a re un abl e to exa mine this phen omenon in mor e det ail because of th e relatively slow ste p cha nge in pr essure at tai na ble with our exper ime ntal apparatus. Thi s activation followin g a rapid change in pressure ha s also been obse rved for th e MscS in in sit u record in gs from gia nt E. coli Sepharose beads to E. coli ceillysates . Anal ysi s of th e material a bsor bed by th e glu ta t hione -Se pha rose beads reveal ed a single major protein band of approximately 40 kDa (F ig. 2A, lan e 3) . After incubation of th e Sepharose-bound fusion protein with thrombin , two additional protein bands of 26 and 17 kDa wer e gen erated , pr esumably representing GST a nd MscL, resp ecti vely (Fig. 2A,lane 4 ). Following thrombin digestion, th e MscL channel protein wa s furth er purified by removal of th e GST portion of th e fusion protein with th e beads fraction ( Fig. 2A,  lane 5 ). Th e molecul ar weight of th ese protein bands corres ponds well with tho se pr edicted from th e DNA se que nce .
Gen eration and Effect of Anti-MscL Polyclonal Antisera -Polyclonal a nti-MscL antisera were gene rate d by inj ecting purified MscL protein into rabbits. Both animal s showed signi ficant a nti-MscL titers se veral week s a fte r inj ecti on , as determined by Western blot a nd ELISA analyses of th e collect ed sera. MscL-sp ecific antibodi es were purified by affinity chromatogr aphy and were used in th e Western blot shown in Fig. 2B. Both fusion protein and MscL showed st rong reactivity with the antibodies (Fig. 2B , lan es 3 a nd 5) , wh erea s no sign ifica nt reactivity was observed with an y other E . coli protein s or GST (F ig. 2B , lan es 1 and 4 ). However, in E. coli cell s st r ongly expressing th e fusion protein, se ve ra l sma ller molecular weight protein bands reacted with th e ant ibodies (Fig. 2B , lane 2 ), which pr esumably represent proteolytic degradation products of the fusion protein. Immune reactions of pr e-sera, ser a following MscL injection , a nd a ffinity -pu rified anti-MscL a nti bodies , against purified MscL protein were a lso exa mined in ELISA a na lys is . Pre-sera showed no significa nt reactivity with MscL protein , wh erea s immune blood exh ibite d st rong reactivity; about ha lf of th e antibody titer wa s recovered a fte r purification over a MscL -affinity column (dat a not shown ).
El ectrophysiologi cal Recording s of Reconstituted MscL Protein-Purified MscL protein wa s recon stituted into lipo som es (prote in :lipid ratio of 1:6000) and obs erv ed to be fun ction a l wh en exa mine d with th e patch-clamp technique (F ig. 3). Single-cha n ne l currents were recorded from excised patch es of liposom e membrane at pip ette pot ential s ranging from -40 mV to + 40 mV and pr essures ranging from -50 to -200 mmHg. No channel activity wa s observed in excise d patches from liposomes not containing MscL protein ( n = 5 patches ). In addit ion, no activity wa s observed in patches from liposomes containing eit her GST-M scL fusion protein (n = 8 patches; pr ot ein :lipid ratio of 1:3000) or GST protein a lone ( n = 4 patches; protein: lipid r atio of 1:5900 ).
Activation of th e MscL by pr essure cea sed followin g removal of th e st imulus as shown in Fi g. 3. As pr essure is increased in small ste ps , the threshold of activation is crossed , a nd th e ch annel a ct ivate d. Furth ermore, if the a pplie d nega tiv e pr essu re is maintained at a constant level, th e chan ne l act ivity in many patches slowly in cr eased with time (Fig. 3). In patches where th e number of act ive channels wa s low, MscL activity occurred as a burst of single openings followed by a lon g inac- Recordings of 20-s duration were obtained from isolated patches ofliposome membrane in response to normal pressure application, and amplitudes were estimated from the current-amplitude histograms. Data are presented as mean ± S.D. from n patches for the following: +20, + 10, -10, -20 mV (n = 7), +30, -30 mV (n = 3), and +40, -40 mV (n = 2).
shown (9». Effects of Antibodies in Patch-Clamp Experiments-Liposomes containing either purified MscL or MscL which had been preincubated in a 1:1 molecular ratio of affinity-purified anti-MscL antibodies for 1 h, were examined. In 11 of the 16 patches examined containing MscL alone, single-channel currents were observed under standard conditions of voltage and pressure. However, in 16 patches examined where MscL was preincubated with anti-MscL antibody, no single-channel opening events were observed at pressures up to -200 mmHg.

DISCUSSION
In this study, we have used a common method of expressing recombinant proteins in E. coli as fusion proteins with GST (14,15) to produce substantial amounts of purified MscL protein. A plasmid expression vector was constructed encoding a hybrid protein with fusion of MscL to the C terminus ofGST separated by a thrombin cleavage site. Induction of the hybrid gene resulted in strong expression of the fusion protein followed by a rapid single-step purification from E. coli cell lysates using glutathione-coated beads. The recombinant MscL protein was further purified by mild detergent extraction following thrombin cleavage of the fusion protein bound to the beads.
Proteolytic digestion of the fusion protein by thrombin resulted in the presence of several amino acid residues at the N terminus of the recombinant protein that are not found in the wild-type MscL protein, as confirmed by N-terminal amino acid sequence analysis. The experiments with the recombinant (Eq.1) spheroplasts (21).
Conductance Measurements of Reconstituted MscL-The conductance of the purified MscL was estimated from the amplitude of the single-channel currents and the applied pipette voltage (Fig. 6). The MscL showed slight rectification at positive pipette voltages. The conductance at negative potentials using a linear regression fit to the data was 3,500 pS and, for positive potentials, was 3,300 pS, with the reversal potential close to zero as expected for this nonselective ion channel (9).
Inhibition of the MscL by Gadolinium-In the present study, reconstituted MscL was inhibited by gadolinium in a reversible manner (Fig. 7). Complete blockade of channel activity by 1.0 mM gadolinium was observed even at negative pressures up to 150 mmHg. At a lower concentration (0.2 ma), inhibition by gadolinium was still observed; however, this inhibition could be reversed by increasing the applied pressure (data not MscL did not indicate any major effects of these additional amino acid residues on channel properties. However, unlike the native MscL protein examined following gel filtration and reconstitution, which does not exhibit rectification (9), at positive pipette voltages a slight rectification was observed with the recombinant channel. At present, we are unable to explain this observation, but one possibility may be that the additional ten amino acids present on the recombinant MscL interfere with the unidirectional passage of ions through the channel pore. With these amino acids present, it is unlikely that the Nterminal portion ofMscL plays a major role in the transduction of mechanical force used for activation of this channel, since the activation pressures for the recombinant MscL were similar to those observed for the native protein reconstituted in liposomes (9).
In the present study, we have used the purified recombinant protein to generate specific polyclonal anti-MscL antibodies which showed strong reactivity with both fusion protein and MscL in Western blot and ELISA analyses. When incubated with MscL protein prior to reconstitution, these antibodies abolished channel activity. These anti-MscL antibodies should enable us to study MscL location in the native E. coli cell envelope, as well as to identify cross-reactive proteins in other organisms.
Purified MscL protein was reconstituted into liposomes and found to be fully functional, exhibiting characteristic conductance and pressure sensitivity, similar to that of the native channel (9). In addition, following incorporation into liposomes, the recombinant channel was blocked by the common inhibitor of mechanosensitive channels, gadolinium (4), at concentrations similar to those reported to inhibit the MscL of E. coli following reconstitution of solubilized native membranes (7,9). Gadolinium appeared to increase the activation threshold of the MscL, suggesting a partial reversal of the inhibition by pressure. However, since in the majority of cases the number of channels observed per patch was relatively high (on average 3 to 6), this result may also reflect that due to the increased open probability, there is an increased likelihood of observing those channels in the patch not inhibited by gadolinium.
The number of channels present in a particular patch of membrane appeared to influence the type of activity displayed by the recombinant protein. Where the number of active channels in the patch was relatively low (1-2), openings occurred as a single burst followed by long inactivation. In the majority of patches where the number of active channels was higher, the MscL exhibited sustained activity, and, furthermore, in many patches channel activity was observed to slowly increase with time. A similar increase in activity of the MscL from native E. coli membranes has been observed, and, typically, channel activity continues to increase with time until the patch ruptures.f Taken together, the results suggest that upon application of negative pressure there may be cooperativity between MscL molecules, either with regard to activation or association of the channel monomers with one another. It is tempting to speculate that the possible mechanism of MscL activation by lipid bilayer tension consists of assembly of pore-forming multimers from dispersed channel monomers in response to mechanical force. A multimeric form (possibly a tetramer) of the functional channel (9) is suggested from the observation that the native channel purified from the E. coli cell envelope has an approximate molecular mass of60-80 kDa compared to that of the monomer of 15 kDa. A possible indication of an association mechanism for MscL activation comes from the results of the polyclonal antibody experiments. When mixed with the channel protein prior to incorporation into liposomes, anti-MscL antibodies prevented any channel activity from being observed. However, another more trivial explanation for the effect of the antibodies may simply be that the MscL protein-antibody complex may not insert into the lipid bilayer in a way which allows channel activation by pressure. Further evidence for interaction between functional MscL derives from the observation that the recombinant MscL was more responsive to rapid steps in pressure than in response to a gradual increase in stimulus. A similar phenomenon has been reported for mechanosensitive channels of hair cells of the turtle and stretch-activated channels of Xenopus oocytes (22). A possible physiological role for this rapid activation may be in providing part of a defense mechanism against rapid changes in osmotic pressure (7).
Activation by pressure of mechanosensitive channels in E. coli can be described by a Boltzmann distribution (8). However, for the recombinant MscL, in many patches continuous application of pressure resulted in an increase in channel activity with time, and, furthermore, where channel number was low, channel inactivation was observed. Therefore, in these experiments, the Boltzmann distribution could only be used as an approximate description of the channel quasi-steady-state activity, since the channels were not truly in an equilibrium state. However, the results for this apparent pressure sensitivity of MscL did show that the reconstitution method decreases the pressure required to activate these channels without altering the activation profile with respect to pressure. A similar lowering in activation pressure threshold has been reported for both MscS and MscL when purified native channels were incorporated into liposomes (9).
In conclusion, we have used a common method for expressing recombinant proteins in E. coli to produce significant amounts of purified MscL protein, and the recombinant channel isolated was found to be fully functional when reconstituted into artificial liposomes. Furthermore, the rapid protein purification method described in this paper will enable us to examine mutagenized MscL proteins and hence explore the role of specific