Selective Extraction and Purification of a Mycobacterial Outer Membrane Protein

Analytical Biochemistry 285, 113–120 (2000) doi:10.1006/abio.2000.4728, available online at http://www.idealibrary.com on

Selective Extraction and Purification of a Mycobacterial Outer Membrane Protein Christian Heinz and Michael Niederweis 1 Lehrstuhl fu¨r Mikrobiologie, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Staudtstrasse 5, 91058 Erlangen, Germany

Received April 13, 2000

MspA forms water-filled channels in the mycolic acid layer of Mycobacterium smegmatis thereby allowing the diffusion of hydrophilic solutes through this permeability barrier into the periplasm. MspA is the first member of a new family of porins and is extremely stable against chemical and thermal denaturation. We developed a purification procedure based on selective extraction of MspA with detergents from whole cells of M. smegmatis at high temperatures. Anion-exchange and size-exclusion chromatography yielded about 230 ␮g apparently pure and highly active MspA per liter of culture. This was a 20-fold increased yield compared to previous purification protocols. Similar amounts of pure MspA were obtained with the detergents isotridecylpolyethyleneglycolether, lauryldimethylamine oxide, and octylpolyethylene oxide indicating that this purification procedure is not restricted to a specific detergent. This study will promote the structural and functional analysis of MspA and might be valuable for the isolation of porins from other mycolic acid-containing bacteria. © 2000 Academic Press

Key Words: porin; cell wall channel; mycolic acid; lipid bilayer; detergent; stability.

The mycobacterial cell envelope is unique among bacteria. Its mycolic acid layer is about twice as thick as the outer membrane of gram-negative bacteria and resembles its function by forming an efficient permeability barrier (1). The diffusion of hydrophilic solutes through this mycobacterial outer membrane is permitted by channel-forming proteins, the porins (2), but is 100- to 1000-fold less efficient compared to Escherichia 1 To whom correspondence should be addressed. Fax: ⫹49/9131/ 85-28082. E-mail: [email protected].

0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

coli (3, 4) causing an intrinsic resistance of mycobacteria to hydrophilic antibiotics. MspA was the first porin identified in Mycobacterium smegmatis and forms a cation-selective, waterfilled channel which is composed of 20-kDa subunits (5). The MspA channel is extremely stable against chemical and thermal denaturation; e.g., the channel-forming activity is not lost after extraction with organic solvents or boiling with 3% sodium dodecyl sulfate (SDS) (5). MspA must form channels of about 10 nm length to span the mycolic acid layer of mycobacteria and, therefore, constitutes the longest channels known to date. These data indicate that MspA is different from the well-characterized porins of gram-negative bacteria (6) and is probably the prototype of a new family of channel-forming proteins. Since MspA appears to be the main channel in M. smegmatis (5), structural and functional studies of MspA would be important for our understanding of how the accessibility of hydrophilic molecules into M. smegmatis is controlled. However, both methods used previously for the preparation of porins from M. smegmatis yielded only a few micrograms of protein (5, 7). Overproduction of MspA in E. coli is possible, but only a small fraction of recombinant MspA was active (5). Therefore, our goal was to improve the purification of porins from M. smegmatis. In this study, we describe an efficient purification procedure based on selective extraction of MspA from whole cells of M. smegmatis with neutral or zwitterionic detergents at high temperatures. It yields pure and highly active MspA in 20-fold increased amounts compared to previous methods. This purification procedure is also applicable to purify other porins from M. smegmatis and should, therefore, promote the structural and functional analysis of this new class of proteins. 113

114

HEINZ AND NIEDERWEIS

MATERIALS AND METHODS

Polyacrylamide Gel Electrophoresis

Chemicals

Protein samples were adjusted to 40 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.0, 3% (w/v) SDS, 8% (w/v) glycerol, 0.1% (w/v) Serva blue G with a fourfold stock solution of loading buffer and incubated at room temperature for 30 min before loading on a denaturing polyacrylamide gel if not stated otherwise. Gels were prepared as described (8, 9) and stained either with Coomassie brilliant blue using standard protocols or with silver (10).

Chemicals were of the highest purity available from Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), or Sigma (Mu¨nchen, Germany). The following detergents were used in this study: SDS from Serva (Heidelberg, Germany); sodium cholate, cetyltrimethylammonium bromide (CTAB), 2 and lauryldimethlyamine oxide (LDAO) from Calbiochem (La Jolla, CA); isotridecylpolyethyleneglycolether (Genapol X-80), noctylthioglucoside, and 3-dodecyldimethylammoniopropane-1-sulfonate (Zwittergent 3-12) from Boehringer (Mannheim, Germany); n-octylpolyoxyethylene (OPOE) from Bachem (Heidelberg, Germany); and polyoxyethylene–sorbitane–monooleate (Tween 80) from Sigma (Mu¨nchen, Germany). Selective Extraction of MspA at High Temperatures For determination of the optimal temperature for extraction of MspA, 10 mg M. smegmatis mc 2155 cells (wet weight) was washed with phosphate-buffered saline (PBS; 100 mM sodium phosphate, pH 7.0, 150 mM NaCl, 0.1 mM EDTA), resuspended in 150 ␮l PG05 buffer (0.5% Genapol, 100 mM Na 2HPO 4/NaH 2PO 4, 0.1 mM EDTA, 150 mM NaCl, pH 6.5), and incubated for 30 min at 30, 40, 50, 60, 70, 80, 90, or 100°C. The samples were cooled on ice for 10 min and centrifuged at 4°C for 10 min. The volume of the supernatants was reduced from 120 to 10 ␮l by evaporation. The proteins were analyzed as described below. Solubilization of MspA by Various Detergents Different detergents were tested in phosphate-based extraction buffers (100 mM Na 2HPO 4/NaH 2PO 4, 0.1 mM EDTA, 150 mM NaCl, pH 6.5). Ten milligrams of M. smegmatis mc 2155 cells (wet weight) was washed with PBS and resuspended in 150 ␮l of extraction buffer containing 0.5% (w/v) SDS (PS05), 0.9% (w/v) cholate (PCH09), 0.1% (w/v) CTAB (PCT01), 0.05% (w/v) LDAO (PLD005), 0.1% (w/v) Zwittergent 3-12 (PZ01), 0.6% (w/v) octylthioglucoside (POT06), 0.5% (w/v) OPOE (POP05), 0.01% (w/v) Genapol (PG001), or 0.5% (w/v) Genapol (PG05). The samples were boiled for 30 min in a water bath, cooled on ice for 10 min, and centrifuged at 4°C for 10 min. The proteins were analyzed as described below.

Large-Scale Purification of MspA Ten grams M. smegmatis cells (wet weight) was washed with PBS, resuspended in 35 ml POP05, and boiled under stirring for 30 min in a water bath. The suspension was cooled on ice for 10 min and centrifuged at 4°C for 15 min at 27,000g. Forty-two milliliters of the supernatant was gently mixed with an equal volume of ice-cold acetone. This mixture was kept on ice for 1 h and centrifuged at 4°C for 15 min at 8000g. The precipitated protein was dissolved in 10 ml 25mM N-(2-hydroxyethyl)piperazine-N⬘-2-ethane sulfonic acid (Hepes), pH 7.5, 10 mM NaCl, 0.5% OPOE (AOP05) and loaded on an anion-exchange column POROS 20HQ with a volume of 1.7 ml (Perseptive Biosystems, Cambridge, MA). After washing the column with 14 ml AOP05, bound proteins were eluted with a gradient from 100% AOP05 to 100% BOP05 (25 mM Hepes, pH 7.5, 2 M NaCl, 0.5% OPOE) over 34 ml. Ninety fractions of 1 ml were collected. The fractions were analyzed using denaturing polyacrylamide gels which were stained with silver. Four fractions with the highest amount of MspA were pooled and the protein was precipitated with acetone as described above. The pellet was dissolved in 600 ␮l AOP05, incubated on ice, and centrifuged at 4°C for 5 min to remove insoluble material. The protein solution was loaded on a gel filtration column Superdex 200 with a volume of 24 ml (Pharmacia, Freiburg, Germany). Proteins were eluted with 48 ml of AOP05 at a flow rate of 0.2 ml/min. Fifty fractions of 1 ml were collected and analyzed using denaturing polyacrylamide gels which were stained with silver. Fractions containing apparently pure MspA were pooled. The protein concentration was determined as described below. Determination of Protein Concentrations

2 Abbreviations used: PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; Hepes, N-(2-hydroxyethyl)piperazine-N⬘-2-ethane sulfonic acid; BCA, bicinchoninic acid; DMSO, dimethyl sulfoxide; CMC, critical micellar concentration; HPLC, highperformance liquid chromatography; MALDI, matrix-assisted laser desorption ionization; see Table 1 for abbreviations of the detergents.

Protein concentrations were determined using bicinchoninic acid (BCA) (11) and bovine serum albumin (BSA) as standard protein, according to the manufacturer’s instruction (BCA protein assay kit, Pierce, Rockford, IL).

PURIFICATION OF A MYCOBACTERIAL PORIN

115

Lipid Bilayer Experiments For analysis of the channel activity of MspA, both compartments of a Teflon chamber were filled with 5 ml of an aqueous solution of 1 M KCl buffered with 10 mM 2-(N-morpholino)ethanesulfonic acid (Mes), pH 6.0. A 1% (w/v) solution of diphytanoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) in n-decane was painted across a circular hole in the wall separating both compartments. The surface area of these holes was 0.1 or 0.5 mm 2. Samples were added after the lipid membrane turned optically black to incident light. Membrane current was measured after application of a transmembrane potential of 10 mV with a pair of silver electrodes. The current was boosted by a current amplifier (427, Keithley) and recorded with a strip chart recorder. The temperature was kept at 20°C throughout the experiment. Image Processing Polyacrylamide gels were dried using the DryEase mini-gel drying system (Novex, San Diego, CA) and scanned (UC840 Max Vision, Umax). The images were imported into Adobe Photoshop 5.0 (Adobe, Seattle, WA) and brightness and the global gradation curve were adjusted to reduce the background. No parts of the gels were changed individually. Protein amounts were determined densitometrically using the program TINA 2.0 (Raytest Isotopenmessgera¨te GmbH, Straubenhardt, Germany). RESULTS

Selective Extraction of the Porin MspA at High Temperatures FIG. 1. Temperature-dependent extraction of MspA from M. smegmatis. Ten milligrams of M. smegmatis cells was extracted with 150 ␮l of a buffer containing 0.5% Genapol (PG05) at different temperatures for 30 min. The arrows indicate the position of MspA. (A) Coomassie-stained denaturing 10% polyacrylamide gel (9). The volume of each extract was reduced to 10 ␮l by evaporation. The samples were incubated at room temperature with 5 ␮l loading buffer for 30 min. Twelve microliters of each PG05 extract was loaded on the gel. Lane M, molecular mass marker (200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4, 6 kDa); lanes 1 to 8, PG05 extracts obtained at 30, 40, 50, 60, 70, 80, 90, or 100°C. (B) Immunoblot analysis. One microliter of each extract was incubated at room temperature with 1 ␮l loading buffer for 30 min. The proteins were separated on a denaturing 8% polyacrylamide gel (8) and blotted onto a PVDF membrane. Proteins were visualized using the MspA antiserum (5) and a chemiluminescence reaction (ECL detection system, Amersham-Pharmacia, Vienna, Austria). Lane M, molecular mass marker (97.4, 68, 46, 31, 20.1, 14.4 kDa); lanes 1 to 3, 2 ␮l of PG05 extract obtained at 30, 40, or 50°C; lanes 4 to 8, 1 ␮l of the PG05 extracts obtained at 60, 70, 80, 90, or 100°C; lane 9, 1 ng MspA. Please note that different volumes of the extracts were loaded on the gel to visualize the small amounts of MspA obtained at low temperatures. (C) Proteins were separated on a denaturing 8% polyacrylamide gel (8). The gel was stained with silver (10). The volume of each extract was reduced to 10 ␮l by evaporation. The samples were incubated at

For preparation of the porin MspA from M. smegmatis we started with extraction of whole cells with detergents to avoid the loss of material which occurs by isolating cell walls using sucrose density gradient centrifugation. In order to improve both yield and purity of preparations MspA we tried to exploit its unusual stability toward denaturation by heat (5). Therefore, 10 mg cells was extracted with 150 ␮l of a phosphate buffer containing 0.5% Genapol (PG05 buffer) at different temperatures for 30 min. At temperatures above 70°C, significant amounts of a 100-kDa protein were selectively extracted from M. smegmatis cells (Fig. 1A).

room temperature with 5 ␮l loading buffer for 30 min. Lane M, molecular mass marker (200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4, 6 kDa); lane 1, 15 ␮l PG05 extract at 30°C; lane 2, 15 ␮l PG05 extract at 40°C; lane 3, 10 ␮l PG05 extract at 50°C; lanes 4 to 8, 4 ␮l of the PG05 extracts at 60, 70, 80, 90, or 100°C; lane 9, 270 ng purified MspA. Please note that different volumes of the extracts were loaded on the gel to visualize the small amounts of MspA obtained at low temperatures.

116

HEINZ AND NIEDERWEIS TABLE 1

Detergents Tested for the Solubilization of MspA Detergent

Class a

CMC b (mM)

c (mM)

Buffer

Dodecyl sulfate, sodium salt (SDS) Cholate, sodium salt Cetyltrimethylammonium bromide (CTAB) 3-Dodecyldimethylammoniopropane-1-sulfonate (Zwittergent 3-12) n-Dodecyldimethylamine oxide (LDAO) n-Octylpolyethylene oxide (OPOE) n-Octyl-␤-D-thioglucoside (OSG) Isotridecylpolyethyleneglycolether (Genapol X-80)

A A C Z Z N N N

7–10 9–15 1 2–4 1–2 7 9 0.06–0.15

16.6 20 2 4 2 14 18 0.26, 9.1

PS05 PCH09 PCT01 PZ01 PLD005 POP05 POT06 PG001, PG05

a b

Detergents used were anionic (A), cationic (C), zwitterionic (Z), and nonionic (N). The CMC values indicated here were those given by the manufacturers at 50 mM NaCl.

This protein had the same electrophoretic mobility as purified MspA and was recognized by a polyclonal rabbit antiserum to purified MspA from M. smegmatis (pAK MspA#813) (5) in an immunoblot experiment (Fig. 1B). The 100-kDa protein was isolated from the Genapol extract obtained at 100°C by preparative gel electrophoresis and showed a very high channel-forming activity in lipid bilayer reconstitution experiments with a main conductance of 4.6 nS identical to that of the MspA channels (5) (data not shown). Heating the 100-kDa protein in 80% dimethyl sulfoxide (DMSO) to 100°C dissociated it into 20-kDa monomers as it was shown for the MspA porin from M. smegmatis (5) (data not shown). In addition, 17 amino acids of the N-terminus were identical to that of MspA (data not shown). These data indicate that the 100-kDa protein is identical to MspA. It is concluded that high-temperature extracts with 0.5% Genapol selectively extract the MspA porin from M. smegmatis. The amount of extracted MspA strongly increased with temperature. The best yield was obtained after extraction at 90 to 100°C (Figs. 1A and 1B). Shortening the extraction time from 30 to 20 or 10 min reduced the yield of MspA (data not shown). To analyze the influence of the cell density on the yield of MspA, 5, 10, 20, or 40 mg cells was extracted with 100 ␮l PG05 buffer. The best extraction efficiencies were obtained with cell densities between 5 and 10% (data not shown). To analyze whether the Genapol extracts of M. smegmatis cells contained minor amounts of other proteins the extracts were analyzed in a denaturing polyacrylamide gel which was stained with silver (Fig. 1C). In this gel, a 50-kDa protein was visible which was coextracted with MspA at higher temperatures. This protein was not recognized by the MspA-specific antiserum. Another band with a slightly lower apparent molecular mass than MspA was detected in the silverstained gel at extraction temperatures of 90 and 100°C (Fig. 1C). This protein was recognized by the MspAspecific antiserum and is presumably another form of MspA with a different electrophoretic mobility as this

band was also observed after overexpression of MspA in E. coli in addition to the predominant form of MspA at about 100 kDa (data not shown). This appears to be a similar phenomenon to that observed for outer membrane proteins of E. coli (12). It should be noted that the apparent molecular mass of the predominant form of MspA is determined from its electrophoretic mobility and varies between 100 and 115 kDa depending on the conditions of gel electrophoresis. Solubilization of MspA by Various Detergents Different detergents were tested to analyze whether the selective extraction of MspA from M. smegmatis cells is specific for Genapol. For this purpose, the extraction conditions were chosen as determined to be optimal for extraction with Genapol. First, all detergents were used in the same phosphate buffer at a concentration estimated to be roughly twofold above their critical micellar concentration (CMC) (Table 1).

FIG. 2. Detergent-dependent extraction of MspA from M. smegmatis. Ten milligrams of M. smegmatis cells was extracted with 150 ␮l of a detergent-containing buffer at 100°C for 30 min. 8.4 ␮l of each extract was incubated at room temperature with 5 ␮L loading buffer for 30 min before loading on a denaturing 10% polyacrylamide gel (9). The gel was stained with silver (10). Lane M, molecular mass marker (200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4, 6 kDa); lane 1, PS05 extract; lane 2, PCH09 extract; lane 3, PCT01 extract; lane 4, PZ01 extract; lane 5, PLD005 extract; lane 6, POP05 extract; lane 7, POT06 extract; lane 8, PG001 extract; lane 9, PG05 extract. See Table 1 for explanation of the buffer abbreviations. The arrow indicates the position of MspA.

PURIFICATION OF A MYCOBACTERIAL PORIN

117

FIG. 3. Large-scale purification of MspA. (A) Flow chart of the purification steps. The total amount of protein was determined after each step using the BCA assay and is indicated on the right side. The protein content of the detergent extract after selective solubilization was determined to 57 mg. This value was marked with a star, because it probably results from a side reaction of the BCA assay. Quantitative image analysis of the Coomassie-stained denaturing polyacrylamide gel (B) using purified MspA as a reference revealed a protein content of about 1.5 mg. (B) Gel electrophoretic analysis of the purification steps. Proteins were separated on a denaturing 10% polyacrylamide gel (9). The gel was stained with Coomassie. Lane M, molecular mass marker (200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4, 6 kDa); lane 1, 40 ␮g protein of the POP05 extract; lane 2, 40 ␮g protein of the sample of lane 1 after precipitation with acetone; lane 3, 4 ␮g protein of the pooled fractions 48 –51 after anion exchange; lane 4, 4 ␮g protein of the sample of lane 3 after precipitation with acetone; lane 5, 4 ␮g protein of the pooled fractions 12–15 after gel filtration. Please note that the indicated protein masses were determined by the BCA assay. (C) Anionexchange chromatography. The solid line represents the absorbance at 280 nm; the dashed line the conductivity. Elution with a linear gradient from 0 to 1 M NaCl caused MspA to elute between 0.48 and 0.74 M NaCl with the peak eluting at 0.57 M NaCl. The pooled fractions (48 –51) are marked with a bracket. 4 ␮g protein of this sample was analyzed by gel electrophoresis (Fig. 3B, lane 3). (D) Size-exclusion chromatography. The solid line represents the absorbance at 280 nm. The proteins were eluted with 48 ml AOP05. The pooled fractions (12–15) are marked with a bracket. 4 ␮g protein of this sample was analyzed by gel electrophoresis (Fig. 3B, lane 5).

Second, M. smegmatis cells were incubated with the corresponding buffers at 100°C for 30 min and at a cell density of 10%. Extraction with the anionic detergents SDS and cholate solubilized many proteins from M. smegmatis (Fig. 2), but only a few channels were observed in lipid bilayer experiments (data not shown). No protein was detected after extraction with the cationic detergent CTAB in silver-stained denaturing polyacrylamide gels and no channel activity was observed (Fig. 2). However, extracts with the neutral detergents octylthioglucoside and OPOE had a high channel-forming activity and contained a 100-kDa protein from M. smegmatis (Fig. 2). The 100-kDa protein from the OPOE extract was purified and showed a very high channel-forming activity in lipid bilayer reconstitution experiments, dissociated into 20-kDa monomers

after heating to 100°C in 80% DMSO and was recognized by a polyclonal rabbit antiserum to purified MspA from M. smegmatis (pAK MspA#813) in immunoblot experiments (data not shown). These results indicated that the selective heat extraction of MspA from M. smegmatis was also possible with the detergents octylthioglucoside and OPOE. Both detergents were effective at concentrations corresponding to a twofold CMC. Equivalent concentrations of Genapol were 70-fold above its CMC (Fig. 2). The zwitterionic detergent LDAO also efficiently extracted MspA. However, other proteins were present in higher amounts compared to extraction with the neutral detergents tested above. The zwitterionic detergent Zwittergent 3-12 seemed to solubilize many proteins but not MspA (Fig. 2). This was confirmed by lipid bilayer experi-

118

HEINZ AND NIEDERWEIS

ments which revealed only a very low channel-forming activity in the Zwittergent extract. Large-Scale Purification of MspA The selective extraction of MspA at high temperatures was the basic step of our new purification procedure and was followed by anion-exchange and sizeexclusion chromatography (Fig. 3A). Ten grams M. smegmatis cells was extracted with OPOE as a detergent in POP05 buffer. The protein concentration of the supernatant was determined to 1.35 mg/ml using the BCA assay. However, only 36 ␮g protein per milliliter was determined by quantitative image analysis of the Coomassie-stained denaturing polyacrylamide gel using purified MspA as a reference (Fig. 3B, lane 1). Most probably, this discrepancy was caused by reducing substances which were released from M. smegmatis during boiling and interfered with the BCA assay. By comparing the staining of the MspA band with that of the whole lane the purity of MspA was estimated to about 85% (Fig. 3B, lane 1). The proteins were precipitated with acetone and were analyzed by PAGE (Fig. 3B, lane 2). Quantitative image analysis revealed that this sample contained about 90% MspA. The concentrated sample was applied to an anion-exchange column. During gradient elution most of the contaminating proteins were separated from MspA, which eluted in a sharp peak at 570 mM NaCl (Fig. 3C). The major part of other proteins was separated from MspA in this step as demonstrated by PAGE analysis of all fractions with a detectable absorption at 280 nm (data not shown). Four MspA-containing fractions were pooled and 4 ␮g protein of this sample was analyzed by PAGE. No contaminating protein was detected in Coomassiestained gels (Fig. 3B, lane 3). The yield was 1.7 mg in agreement with quantitative image analysis using purified MspA as a reference. This indicated that the substance which interfered with the BCA assay after the extraction step was separated from MspA. The proteins were precipitated with acetone and were analyzed by PAGE (Fig. 3B, lane 4). Since silver-stained gels indicated a minor contamination of MspA with proteins of a lower molecular mass (data not shown), we used size-exclusion chromatography (Superdex 200) as the last purification step (Fig. 3A). Native MspA eluted in the second peak after 12.5 ml (Fig. 3D). The large peak at 23 ml elution volume contained only traces of MspA and presumably consisted of detergent micelles. Four MspA-containing fractions were pooled and 4 ␮g protein of this sample was analyzed by PAGE. No contaminating protein was detected in Coomassiestained gels (Fig. 3B, lane 5). The yield was 700 ␮g in agreement with quantitative image analysis using purified MspA as a reference. In addition, analysis of 1 ␮g of this sample did not reveal any contamination with

FIG. 4. Single channel analysis of purified MspA in lipid bilayer experiments. (A) Single-channel recordings of a diphytanoylphosphatidylcholine membrane in the presence of 0.01 ng/ml purified MspA. Protein solutions were added to both sides of the membranes. Data were collected from at least three different membranes. Mainly, conductance steps of 4.6 nS were observed. (B) Analysis of the probability P of a conductance step G for 197 single-channel events. The average single-channel conductance was 4.6 nS for the maximum consisting of 102 single-channel events.

other proteins in silver-stained denaturing polyacrylamide gels (data not shown) indicating that MspA was purified to apparent homogeneity. This result is in agreement with other analytical methods such as HPLC, immunodetection, MALDI mass spectrometry, or N-terminal sequencing by Edman degradation which did not reveal any heterogeneity of MspA. The channel-forming activity of purified MspA was analyzed in lipid bilayer experiments. Addition of 0.1 ng MspA rapidly increased the conductance of a diphytanoylphosphatidylcholine membrane by many orders of magnitude. The concentration of MspA was 10 pg/ml in the bilayer chamber indicating a high specific activity of the purified protein exceeding that determined for MspA purified after extraction with organic solvents about 10-fold (5). The majority of the channels had a conductance of 4.6 nS (Fig. 4) in agreement with the channel activity determined for the detergent extracts of whole cells of M. smegmatis and of MspA purified using organic solvents for extraction (5). Thus, about 230 ␮g apparently pure MspA per liter culture was obtained using the new purification procedure. This corresponds to a 20-fold increased yield compared to the previously published method (5). DISCUSSION

The low amount of available protein has impaired structural and functional studies of mycobacterial porins. In this study, we describe a new purification procedure for MspA, a porin of M. smegmatis (5). The initial high-temperature extraction of whole cells of M. smegmatis with appropriate detergents yielded a protein extract consisting of 85% MspA in its 100-kDa form indicating the high selectivity of this extraction

PURIFICATION OF A MYCOBACTERIAL PORIN

step. Although some protocols for the selective extraction of porins of gram-negative bacteria exist, most of them require isolation of the cell envelopes and sequential extraction with different detergents (13, 14). Temperatures of 80 to 100°C were necessary to solubilize MspA to a significant extent. These conditions exploit the extraordinary thermostability of MspA and are not applicable to purification of porins from gram-negative bacteria, since they dissociate into inactive monomers at temperatures above 55 to 70°C (15, 16). Selective extraction of MspA at high temperatures from whole cells of M. smegmatis was achieved with several nonionic and zwitterionic detergents allowing to purify MspA with an appropriate detergent instead of a timeconsuming and often difficult exchange of the detergent. Porins from mycolic acid-containing gram-positive bacteria such as Corynebacterium glutamicum (17), M. smegmatis (5), Mycobacterium bovis (18), and Rhodococcus erythropolis (19) were previously extracted with a mixture of chloroform and methanol. This technique provided a certain selectivity for the porins, eliminated the need for preparation of the cell wall using sucrose density gradients, and was, therefore, a significant progress compared to the first purification protocols (2, 7). However, it yielded a mixture of few proteins at a rather low yield; e.g., we never obtained more than 10 ␮g MspA per liter of culture of M. smegmatis. Furthermore, completion of the extraction step including ether precipitation took 24 h (17). Sequential extraction of isolated cell walls of these organisms with different detergents was used as an alternative method, but resulted in nonspecific extraction of many cell wall proteins. Porin purification was difficult (18, 20, 21), because it represented only a small fraction of the total protein in the extract. In contrast, selective solubilization of MspA by appropriate detergents from whole cells at high temperatures cut the time from 24 to 1.5 h and avoided the use of organic solvents. The subsequent anion-exchange chromatography removed most of the contaminating proteins. The high concentration of 570 mM NaCl needed for elution of MspA indicated a strong affinity of the quaternized polyethyleneimine material for the porin in the presence of detergents. The final size-exclusion chromatography yielded about 230 ␮g apparently pure and desalted MspA per liter of culture. This was a 20-fold increased yield compared to the purification of MspA using extraction by organic solvents and preparative gel electrophoresis (5). Since both chromatographic steps were finished in less than 4 h, purification of MspA could be completed in 1 day. We purified MspA with the detergents Genapol, LDAO, and OPOE using this method and obtained similar amounts of pure protein. This indicated that our method is not

119

restricted to a specific detergent. These results show that this purification procedure is far superior to all methods previously used for purification of porins from mycobacteria and other mycolic acid-containing bacteria. It was shown that M. smegmatis contains at least three DNA fragments with a high homology to mspA (5), but it is not known whether porins other than MspA are expressed in M. smegmatis. Although, therefore, a contamination of purified MspA with other very similar porins cannot be totally excluded, all analytical methods used so far like gel electrophoresis, HPLC, single-channel conductance measurements, immunodetection, MALDI mass spectrometry, or N-terminal sequencing by Edman degradation did not reveal any heterogeneity of MspA prepared by the protocol described in this study. The procedure for purification of MspA might be valuable also for the preparation of other porins from mycolic acid-containing bacteria (17, 19 –21). A certain stability of the porins against denaturation by heat appears to be essential, because significant amounts of MspA were only solubilized at temperatures above 70°C. The 0.7 nS porin of M. tuberculosis is heat-sensitive (22) and could, therefore, not be purified using the method described in this study (unpublished data). However, the shorter chain length of the mycolic acids of nocardia, corynebacteria, and rhodococci compared to mycobacteria might decrease the critical solubilization temperature and might facilitate the application of the purification procedure to porins of these bacteria. In conclusion, the purification procedure described here is rapid and efficient and yields pure and highly active MspA. This should promote the structural and functional analysis of MspA which is the prototype of a new family of channel-forming proteins with extraordinary biochemical and biophysical properties. ACKNOWLEDGMENTS The generous support by Dr. Wolfgang Hillen is gratefully acknowledged. We thank Dr. Xiyuan Bai for technical help and Dr. Harald Engelhardt and Dr. Christian Berens for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (NI 412/2-1).

REFERENCES 1. Brennan, P. J., and Nikaido, H. (1995) Annu. Rev. Biochem. 64, 29 – 63. 2. Trias, J., Jarlier, V., and Benz, R. (1992) Science 258, 1479 – 1481. 3. Jarlier, V., and Nikaido, H. (1990) J. Bacteriol. 172, 1418 –1423. 4. Chambers, H. F., Moreau, D., Yajko, D., Miick, C., Wagner, C., Hackbarth, C., Kocagoz, S., Rosenberg, E., Hadley, W. K., and

120

5.

6. 7. 8. 9. 10. 11.

12. 13.

HEINZ AND NIEDERWEIS

Nikaido, H. (1995) Antimicrob. Agents Chemother. 39, 2620 – 2624. Niederweis, M., Ehrt, S., Heinz, C., Klo¨cker, U., Karosi, S., Swiderek, K. M., Riley, L. W., and Benz, R. (1999) Mol. Microbiol. 33, 933–945. Schulz, G. E. (1996) Curr. Opin. Struct. Biol. 6, 485– 490. Trias, J., and Benz, R. (1994) Mol. Microbiol. 14, 283–290. Laemmli, U. K. (1970) Nature 227, 680 – 685. Scha¨gger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368 –379. Morrissey, J. H. (1981) Anal. Biochem. 117, 307–310. Smith, P. K., Krohn, I., R., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76 – 85. Heller, K. B. (1978) J. Bacteriol. 134, 1181–1183. Chevalier, G., Duclohier, H., Thomas, D., Shechter, E., and Wroblewski, H. (1993) J. Bacteriol. 175, 266 –276.

14. Exner, M. M., Doig, P., Trust, T. J., and Hancock, R. E. (1995) Infect. Immun. 63, 1567–1572. 15. Markovic-Housley, Z., and Garavito, R. M. (1986) Biochim. Biophys. Acta 869, 158 –170. 16. Engelhardt, H., Gerbl-Rieger, S., Krezmar, D., Schneider-Voss, S., Engel, A., and Baumeister, W. (1990) J. Struct. Biol. 105, 92–102. 17. Lichtinger, T., Burkovski, A., Niederweis, M., Kramer, R., and Benz, R. (1998) Biochemistry 37, 15024 –15032. 18. Lichtinger, T., Heym, B., Maier, E., Eichner, H., Cole, S. T., and Benz, R. (1999) FEBS Lett. 454, 349 –355. 19. Lichtinger, T., Reiss, G., and Benz, R. (2000) J. Bacteriol. 182, 764 –770. 20. Riess, F. G., Lichtinger, T., Yassin, A. F., Schaal, K. P., and Benz, R. (1999) Arch. Microbiol. 171, 173–182. 21. Riess, F. G., Lichtinger, T., Cseh, R., Yassin, A. F., Schaal, K. P., and Benz, R. (1998) Mol. Microbiol. 29, 139 –150. 22. Kartmann, B., Stenger, S., and Niederweis, M. (1999) J. Bacteriol. 181, 6543– 6546.