Biochimica et Biophysica Acta, 493 (1977) 210-215 © Elsevier/North-Holland Biomedical Press BBA 37681 P U R I F I C A T I O N OF P R O T E I N A, AN O U T E R M E M B R A N E C O M P O N E N T M I S S I N G I N E S C H E R I C H I A COLI K-12 ornpA M U T A N T S
TUU-JYI CHAI and JOHN FOULDS* National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, 9000 Rockville Pike, Building lO/Room 9N-119, Bethesda, Md. 20014 (U.S.A.) (Received December 15th, 1976)
SUMMARY Outer membrane materials prepared from an Escherichia coil ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA + strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA + strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II* or B* purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II* or B* that is difficult to solubilize is identical with the more readily solubilized fraction.
INTRODUCTION Recent work in several laboratories has identified a number ofEscherichia coli mutants that are missing a single major outer membrane protein. These mutants have been designed tolG, selected as tolerant to colicin L-JF246 [1], con, selected as defective as a recipient in conjugation with certain donors [2], and tut, selected as resistant to bacteriophage TulI [3]. All three loci, tolG, con, and tut are located between pyrl) andfabA [4] on the genetic map, although only one (tolG) is listed on the latest map of the E. coli K-12 chromosome [5]. Recent studies by Manning et al. [6] and by Henning and coworkers [3] have demonstrated that tolG, con, and tut mutants are defective in the structural gene for the outer membrane protein missing in these strains. Since it is likely that all three types of mutants are defective in the same locus, the locus has been redesignated ompA as the structural gene for outer membrane protein A [7]. Outer membrane protein A has been purified by two groups [8, 9]. Each began with a cell envelope preparation partially solubilized with either a combination of the detergent Triton X-100 and E D T A [8] or 0.5 ~ SDS [9]. These treatments apparently * To whom correspondence should be addressed. Abbreviation: SDS; sodium dodecyl sulfate.
211 solubilized only a fraction of the total outer membrane protein present. There remained the question of whether the soluble and insoluble fractions of outer membrane protein A were identical. In this report we describe the purification in high yield, and characterization of outer membrane protein A from cell envelope preparations in which the proteins were completely solubilized. We found purified outer membrane protein A was identical with protein II* [8] and with protein B* [9]. Further, we found no difference between the more readily extracted fraction of outer membrane protein A (soluble) and that which remained insoluble. MATERIALS AND METHODS Bacterial strain and media. E. coli K-12 strain JF404 was grown in Protease Peptone No. 3 (Difco), Beef Extract (Difco) medium [1] to a concentration of 5 x 108 cells/ml and the cells collected by centrifugation. After washing the cell pellet in 20 volumes of saline, the cells were frozen at --20 °C. The frozen cells were thawed, resuspended in 0.05 M Tris. HC1, pH 7.8, and disrupted by passage through a French pressure cell as previously described [10]. The broken cell suspension was centrifuged at 1000 × g for 20 min to remove the few remaining intact cells. The supernatant solution was then centrifuged at 50 000 × g for 2 h and the pellet, containing primarily cell envelope material, resuspended in 0.05 M Tris. HCI, pH 7.8, and washed twice by centrifugation. The washed material was resuspended in a small amount of buffer at a concentration of about 10 mg/ml protein and stored at --20 °C prior to use. Isolation o f outer membrane protein A. The method we used for the purification of outer membrane protein A by preparative electrophoresis on polyacrylamide gels was suggested to us by Dr. R. Poyton. Discontinuous polyacrylamide slab gels (3.0 mm thick) containing SDS were prepared in a Bio-Rad Model 220 apparatus according to the method of Neville [11]. About 3.5 mg of cell envelope protein in 350#1 was first heated at 100 °C for 2 rain in the presence of 1 ~ SDS, applied in a single well that extended nearly the entire edge to edge distance at the top of the slag gel and electrophoresed for about 16 h until the outer membrane protein A was about 1-2 cm from the bottom of the running gel. This meant more rapid migrating proteins were electrophoresed off the gel and ensured the maximum separation of outer membrane protein A from proteins with similar electrophoretic mobilities. Next, the slab gel was carefully removed and about 1.5 cm cut from each side with a special knife that notched the gel for marking (see Fig. la). The two edge pieces were stained with Coomassie Brilliant Blue G, destained in circulating 1 0 ~ acetic acid, shrunk in 5 0 ~ methanol to their original size, and matched with the unstained portion of the gel, During this time the main portion of the slab gel was stored at 2 °C. It required about 8-10 h for the entire staining, destaining, and shrinking procedure. Control experiments showed little diffusion of proteins in the gel stored at 2°C even after 20 h. The portion of the unstained gel containing outer membrane protein A could be easily identified after the stained edge strips were matched (Fig. la). This portion of the gel was cut out and then inserted into a 0.5 × 13 cm glass tube. One end of this tube had been sealed with about 1 cm of running gel [10] as a cushion. The sealed end was tightly fitted with a short length of dialysis tubing containing about 0.5 ml of lower
212
B
A
Jbe D
~Z~ZZ~ZZZZZ~ZZZZZ~Z~ZS~ZZSZZZZZ
"--Outer membrane protein A
| hion bag
Fig. 1. Isolation of outer membrane protein A by polyacrylamide slab gel electrophoresis. (A) Photograph of stained edge strips and diagram of unstained portion of slab gel. q-heedge pieces have been matched by means of notches and the portion of the unstained gel containing the outer membrane protein A has been indicated by dashed lines. This portion will be cut out and inserted into the tube depicted in part B. (B) Diagram of tube with the gel strip containing outer membrane protein A prior to electrophoresis of the protein into the buffer contained within the dialysis bag. reservoir electrophoresis buffer and, to ensure a good seal, the overlap between the dialysis tubing and glass was wrapped with parafilm (see Fig. lb). It is important to exclude air bubbles from the buffer within the dialysis bag. Outer membrane protein A was next electrophoresed off the gel strip, through the cushion of running gel and into the buffer contained within the dialysis bag. This required about 18 h at 0.5 mA/tube. A portion of the material in the dialysis bag was examined to insure its purity by slab gel electrophoresis and the remaining material lyophilized.
Analytical methods. Protein was determined by the method of Lowry et al. [12] with bovine serum albumin as a standard. Analytical gel electrophoresis was carried out using three methods, alkaline urea gel [13], Bragg-Hou gel system [14] and the discontinuous buffer system of Neville [11]. The orcinol procedure assay described by Hewitt [15] was used to determine the total neutral sugar using D-galactose and Dmannose as standards. Phosphorous was measured according to the method of Ames and Dubin [16]. Quantitation of SDS was performed as described by Ray et al. [17]. For amino acid analysis, an adequate amount of lyophilized, SDS-free protein was dissolved in 1 ml of constant boiling HCI, sealed under vacuum, and hydrolyzed at 110 °C for 24, 48, or 72 h. The hydrolyzed material was chromatographed on a Beckman model 120C Automatic Amino Analyzer. N-Terminal amino acid was determined by the procedure by Weiner et al. [18]. RESULTS
Yield of outer membrane protein A. Using the dual slab gel electrophoresis apparatus, 7 mg of total cell envelope protein was applied and 0.5 mg of pure outer
213 membrane protein A recovered after the acetone/water extraction (see below). Assuming outer membrane protein A accounts for about 10 ~o of the total cell envelope protein [10], the yield was about 70~o. We found that prolonged electrophoresis of outer membrane protein A from the gel strips into the dialysis bags resulted in significant loss of protein. For example, using [3H]leucine-labeled cell envelope material, increasing electrophoresis time from 18 to 34 h resulted in a 30~o loss of labeled material from the dialysis bag. The material from 16 slab gels was pooled and examined electrophoretically at concentrations that approached the limits of the gel. Only a single band of material staining with Coomassie Brilliant Blue G was visualized in the three gel systems described in Materials and Methods.
Composition of outer membrane protein A. The lyophilized material containing outer membrane protein A was extracted with acetone/water (5:1, v/v) and the extracts analyzed for protein, phosphorus, carbohydrate and SDS. No detectable protein was extracted. Table I shows that the lyophilized material contained significant amounts of phosphorus, SDS and carbohydrate and that the phosphorus and SDS were removed by three acetone/water extracts. The residual carbohydrate was not removed by five additional extractions. The final residue contained about 2 mol of sugar/mol of 33 000 dalton protein. TABLE I EXTRACTION OF SDS, PHOSPHORUS AND CARBOHYDRATE FROM POOLED MATERIAL CONTAINING OUTER MEMBRANE PROTEIN A Fraction
Volume (ml)
SDS (g/100 ml)
Phosphorus (nmol/ml)
Carbohydrate (nmol/ml)
Original pooled material a 1st acetone extract 2nd acetone extract 3rd acetone extract Final pellet, resuspended"
5.0 5.0 5.0 5.0 5.0
1.57 1.33 0.06 0.005 0.0001
375 351 15
TUU-JYI CHAI and JOHN FOULDS* National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, 9000 Rockville Pike, Building lO/Room 9N-119, Bethesda, Md. 20014 (U.S.A.) (Received December 15th, 1976)
SUMMARY Outer membrane materials prepared from an Escherichia coil ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA + strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA + strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II* or B* purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II* or B* that is difficult to solubilize is identical with the more readily solubilized fraction.
INTRODUCTION Recent work in several laboratories has identified a number ofEscherichia coli mutants that are missing a single major outer membrane protein. These mutants have been designed tolG, selected as tolerant to colicin L-JF246 [1], con, selected as defective as a recipient in conjugation with certain donors [2], and tut, selected as resistant to bacteriophage TulI [3]. All three loci, tolG, con, and tut are located between pyrl) andfabA [4] on the genetic map, although only one (tolG) is listed on the latest map of the E. coli K-12 chromosome [5]. Recent studies by Manning et al. [6] and by Henning and coworkers [3] have demonstrated that tolG, con, and tut mutants are defective in the structural gene for the outer membrane protein missing in these strains. Since it is likely that all three types of mutants are defective in the same locus, the locus has been redesignated ompA as the structural gene for outer membrane protein A [7]. Outer membrane protein A has been purified by two groups [8, 9]. Each began with a cell envelope preparation partially solubilized with either a combination of the detergent Triton X-100 and E D T A [8] or 0.5 ~ SDS [9]. These treatments apparently * To whom correspondence should be addressed. Abbreviation: SDS; sodium dodecyl sulfate.
211 solubilized only a fraction of the total outer membrane protein present. There remained the question of whether the soluble and insoluble fractions of outer membrane protein A were identical. In this report we describe the purification in high yield, and characterization of outer membrane protein A from cell envelope preparations in which the proteins were completely solubilized. We found purified outer membrane protein A was identical with protein II* [8] and with protein B* [9]. Further, we found no difference between the more readily extracted fraction of outer membrane protein A (soluble) and that which remained insoluble. MATERIALS AND METHODS Bacterial strain and media. E. coli K-12 strain JF404 was grown in Protease Peptone No. 3 (Difco), Beef Extract (Difco) medium [1] to a concentration of 5 x 108 cells/ml and the cells collected by centrifugation. After washing the cell pellet in 20 volumes of saline, the cells were frozen at --20 °C. The frozen cells were thawed, resuspended in 0.05 M Tris. HC1, pH 7.8, and disrupted by passage through a French pressure cell as previously described [10]. The broken cell suspension was centrifuged at 1000 × g for 20 min to remove the few remaining intact cells. The supernatant solution was then centrifuged at 50 000 × g for 2 h and the pellet, containing primarily cell envelope material, resuspended in 0.05 M Tris. HCI, pH 7.8, and washed twice by centrifugation. The washed material was resuspended in a small amount of buffer at a concentration of about 10 mg/ml protein and stored at --20 °C prior to use. Isolation o f outer membrane protein A. The method we used for the purification of outer membrane protein A by preparative electrophoresis on polyacrylamide gels was suggested to us by Dr. R. Poyton. Discontinuous polyacrylamide slab gels (3.0 mm thick) containing SDS were prepared in a Bio-Rad Model 220 apparatus according to the method of Neville [11]. About 3.5 mg of cell envelope protein in 350#1 was first heated at 100 °C for 2 rain in the presence of 1 ~ SDS, applied in a single well that extended nearly the entire edge to edge distance at the top of the slag gel and electrophoresed for about 16 h until the outer membrane protein A was about 1-2 cm from the bottom of the running gel. This meant more rapid migrating proteins were electrophoresed off the gel and ensured the maximum separation of outer membrane protein A from proteins with similar electrophoretic mobilities. Next, the slab gel was carefully removed and about 1.5 cm cut from each side with a special knife that notched the gel for marking (see Fig. la). The two edge pieces were stained with Coomassie Brilliant Blue G, destained in circulating 1 0 ~ acetic acid, shrunk in 5 0 ~ methanol to their original size, and matched with the unstained portion of the gel, During this time the main portion of the slab gel was stored at 2 °C. It required about 8-10 h for the entire staining, destaining, and shrinking procedure. Control experiments showed little diffusion of proteins in the gel stored at 2°C even after 20 h. The portion of the unstained gel containing outer membrane protein A could be easily identified after the stained edge strips were matched (Fig. la). This portion of the gel was cut out and then inserted into a 0.5 × 13 cm glass tube. One end of this tube had been sealed with about 1 cm of running gel [10] as a cushion. The sealed end was tightly fitted with a short length of dialysis tubing containing about 0.5 ml of lower
212
B
A
Jbe D
~Z~ZZ~ZZZZZ~ZZZZZ~Z~ZS~ZZSZZZZZ
"--Outer membrane protein A
| hion bag
Fig. 1. Isolation of outer membrane protein A by polyacrylamide slab gel electrophoresis. (A) Photograph of stained edge strips and diagram of unstained portion of slab gel. q-heedge pieces have been matched by means of notches and the portion of the unstained gel containing the outer membrane protein A has been indicated by dashed lines. This portion will be cut out and inserted into the tube depicted in part B. (B) Diagram of tube with the gel strip containing outer membrane protein A prior to electrophoresis of the protein into the buffer contained within the dialysis bag. reservoir electrophoresis buffer and, to ensure a good seal, the overlap between the dialysis tubing and glass was wrapped with parafilm (see Fig. lb). It is important to exclude air bubbles from the buffer within the dialysis bag. Outer membrane protein A was next electrophoresed off the gel strip, through the cushion of running gel and into the buffer contained within the dialysis bag. This required about 18 h at 0.5 mA/tube. A portion of the material in the dialysis bag was examined to insure its purity by slab gel electrophoresis and the remaining material lyophilized.
Analytical methods. Protein was determined by the method of Lowry et al. [12] with bovine serum albumin as a standard. Analytical gel electrophoresis was carried out using three methods, alkaline urea gel [13], Bragg-Hou gel system [14] and the discontinuous buffer system of Neville [11]. The orcinol procedure assay described by Hewitt [15] was used to determine the total neutral sugar using D-galactose and Dmannose as standards. Phosphorous was measured according to the method of Ames and Dubin [16]. Quantitation of SDS was performed as described by Ray et al. [17]. For amino acid analysis, an adequate amount of lyophilized, SDS-free protein was dissolved in 1 ml of constant boiling HCI, sealed under vacuum, and hydrolyzed at 110 °C for 24, 48, or 72 h. The hydrolyzed material was chromatographed on a Beckman model 120C Automatic Amino Analyzer. N-Terminal amino acid was determined by the procedure by Weiner et al. [18]. RESULTS
Yield of outer membrane protein A. Using the dual slab gel electrophoresis apparatus, 7 mg of total cell envelope protein was applied and 0.5 mg of pure outer
213 membrane protein A recovered after the acetone/water extraction (see below). Assuming outer membrane protein A accounts for about 10 ~o of the total cell envelope protein [10], the yield was about 70~o. We found that prolonged electrophoresis of outer membrane protein A from the gel strips into the dialysis bags resulted in significant loss of protein. For example, using [3H]leucine-labeled cell envelope material, increasing electrophoresis time from 18 to 34 h resulted in a 30~o loss of labeled material from the dialysis bag. The material from 16 slab gels was pooled and examined electrophoretically at concentrations that approached the limits of the gel. Only a single band of material staining with Coomassie Brilliant Blue G was visualized in the three gel systems described in Materials and Methods.
Composition of outer membrane protein A. The lyophilized material containing outer membrane protein A was extracted with acetone/water (5:1, v/v) and the extracts analyzed for protein, phosphorus, carbohydrate and SDS. No detectable protein was extracted. Table I shows that the lyophilized material contained significant amounts of phosphorus, SDS and carbohydrate and that the phosphorus and SDS were removed by three acetone/water extracts. The residual carbohydrate was not removed by five additional extractions. The final residue contained about 2 mol of sugar/mol of 33 000 dalton protein. TABLE I EXTRACTION OF SDS, PHOSPHORUS AND CARBOHYDRATE FROM POOLED MATERIAL CONTAINING OUTER MEMBRANE PROTEIN A Fraction
Volume (ml)
SDS (g/100 ml)
Phosphorus (nmol/ml)
Carbohydrate (nmol/ml)
Original pooled material a 1st acetone extract 2nd acetone extract 3rd acetone extract Final pellet, resuspended"
5.0 5.0 5.0 5.0 5.0
1.57 1.33 0.06 0.005 0.0001
375 351 15
