The Major Proteins of the Escherichia coli Outer Cell Envelope Membrane

Transcription

1 Eur. J. Biochcm (1975) The Major Proteins of the Escherichia coli Outer Cell Envelope Membrane Preparative Isolation of All Major Membrane Proteins Ingrid HINDENNACH and UIf HENNING Max-Planck-Institut fur Biologie. Tubingen (Received July 12, 1975) A procedure is described that from one batch of cells allows the isolation of all major proteins of the outer cell envelope membrane of Escherichiu coli B/r. The method involves differential extraction of cell envelopes with ionic and non-ionic detergents with and without Mg2+ present, and the proteins are finally separated by molecular sieve chromatography in the presence of sodium dodecylsulfate. From 200 g cell paste in ten days (including the five days chromatography) x 120 mg protein I (molecular weight x 38000), = 110 mg protein 11* (molecular weight = 33000), z 50 mg protein 111 (molecular weight x 17000), and z 30 mg protein IV (molecular weight z 7000) are obtained in pure state, and these yields are near the expected ones assuming quantitative recoveries. Protein 11* is a heat-modifiable protein (perhaps due to complete unfolding and/or binding of sodium dodecylsulfate only at higher temperatures), and the isolated protein is completely in its unmodified form. Protein IV, Braun s lipoprotein, in the cell envelope exists in two forms, one covalently bound to the murein layer and the other not. The isolated protein IV represents the free form of the protein that so far had not been isolated; its protein part does not differ substantially from that of the bound form. The outer cell envelope membrane of Escherichiu coli, and very likely of gramnegative bacteria in general, is faily unique in that it contains only a few species of proteins in rather high concentrations (e.g.[l- 5]), and most other proteins of this membrane are present at much lower amounts. These major proteins cannot be removed from the membrane other than by the use of detergents [4-61, and all of them are either completely or partially resistant to tryptic digestion as long as they are associated with their membrane [1,4,7] or the murein [5]. Also, of all proteins of the outer membrane it is only this set of proteins that constitutes the protein part of E. cofi ghosts [7]. It appears that they may be classified as typical integral membrane proteins. These proteins obviously are of interest not only per se but also concerning questions on the architecture of the outer membrane, its functions and its assembly. In addition, it has recently been shown that mutants can be selected for, that lack one or the other or several of these proteins [8-111; also, mutants have become available which harbor altered forms of one of the proteins [12]. Since there are not many and since they are produced in large amounts it may be that of all biological membranes fully amenable to experiments asking for structure, function and assembly this outer membrane may constitute a relatively simple system. For only one of these proteins, the lipoprotein of molecular wcight about 7000 studied by Braun and collaborators [13], has it been proven beyond doubt that it constitutes a single polypeptide chain since its (rather unusual) primary structure has been elucidated. The others constitute a set of polypeptide chains in a molecular weight range between about and (see below). It has so far not become clear how many different species of these polypeptides exist, and in several cases where one or the other of the proteins has been isolated all evidence availablc does not allow the strict conclusion that a single, homogeneous polypeptide has been studied (for further details see Discussion and the following communicat ion). We have attempted to clarify the situation, and here we describe the separation and isolation of the proteins in question. Evidence for or against homogeneity of the isolated polypeptides will be presented in the accompanying communication.

2 208 Escherichin coli Cell Wall Proteins MATERIALS AND METHODS Cells For purification work E. coli B/r was obtained commercially from Merck (Darmstadt). It was grown aerobically at 37 C in a medium containing 27; glucose; 0.5 % casein peptone; 0.5x meat peptone; 0.5% yeast extract; 0.3 % (NH4),HP04; 0.02% MgS04; 33 mm phosphate buffer, ph 6.8. The cells were harvested at one half of the logarithmic phase of growth. PROTEIN PURIFICATION Electrophoresis The term electrophoresis is used exclusively for dodecylsulfate-polyacrylamide gel electrophoresis. Two methods were employed: (a) (in tubes with a phosphate buffer system) was as described before [7]; and (b) (slab gel with a Tris/HCl buffer system), was according to Studier [14] as detailed for bacterial membranes by Ames [15]. Apparent molecular weights were determined with method (b), and otherwise according to Weber and Osborn [16]. Step 1 : Envelope Fraction To 200 g cell paste were added 400 g aluminum oxide (Alcoa Alumina A-305, Alcoa Deutschland GmbH, Frankfurt), 50 ml 0.01 M Tris/HCl, ph 7.5, containing 0.05 % mercaptoethanol, and 2 mg DNAase (Worthington). The mass was ground at 4 C for 20 rnin in a motor-driven mortar, 150 ml of the same Tris/HCl buffer were added and grinding was continued for another 2 min. The homogenate was centrifuged for 3 min at 1000 x g (removal of A1,03). The pellet was resuspended with 200 ml Tris/HCl buffer and again centrifuged for 3 rnin at 1000 x g. The combined supernatants were left for 30 rnin at 4"C, centrifuged for 60min at 200OOxg, and the supernatant was discarded. The electrophoretic profile of the envelope fraction is shown in Fig. 1. Step 2: Extraction with +- Mg2 Sodiuin Dodecylsuljate The sediment was suspended with 300 ml 0.01 M Tris/HCl, ph 7.5, containing 4% sodium dodecylsulfate, 0.05 % mercaptoethanol, and 0.5 mm MgC1, and held with stirring at 30 C for 30 min. The suspension was then centrifuged for 50 rnin at x g. The sediment was treated identically once more, and the supernatants were discarded. The electrophoretic profiles of the two supernatants are shown in Fig. 1. There is some loss of proteins II*, 111, and IV (second supernatant), the nature of this protein 11* is discussed in the Results section. Step 3: Extraction with EDTA - Sodium Dodecylsulfate Sediment from step 2 was suspended with 100 ml 0.01 M Tris/HCl, ph 7.5, containing 2% sodium dodecylsulfate, 0.05 % mercaptoethanol, and 5 mm EDTA. The suspension was stirred for 30 rnin at 30 "C and centrifuged for 60 min at xg. The sediment was treated once more with 75 ml extraction buffer in the same way, and the supernatants were combined. Fig. 1 shows the electrophoretic profiles of the combined supernatants and the sediment. Step 4: Trypsin Treatment Sediment from step 3 was suspended with 100 ml water and 900 ml acetone were added. Insoluble material was allowed to settle at 4 C and the precipitate was washed two times with 500 ml 905< acetone in order to remove the sodium dodecylsulfdte. The washed material was lyophilized from aqueous suspension (dry weight: g). It was suspended (50 mg/ml) with 0.02 M Tris/HCl, ph 9, and trypsin was added at 200 pg/ml. The suspension was left at room temperature for about 12 h and then centrifuged for 30 rnin at x g. The supernatant was discarded and the sediment washed, by the same centrifugation, three times each with 1 volume Tris/HCl, ph 9, once with water, and then lyophilized (dry weight about 2 g). The protein composition of this trypsinized material is shown in Fig. 4. Step 5a: Chromutography on Biogel P150 (Proteins I, II, III) Dry material from step 4a (2 g) was suspended with 100 ml 0.01 M Tris/HCI, ph 7.5. containing 2"4 sodium dodecylsulfdte and 5 mm EDTA. The suspension was kept for 5 min in a boiling-water bath, and insoluble material was removed by centrifugation for 60 rnin at xg. The clear supernatant was applied to a jacketed 10 x 80-cm Biogel P150 column (gel bed volume 7 1) and chromatographed at 30 C with 0.01 M Tris/HCl, ph 7.5, containing 1.5 yg sodium dodecylsulfate, 5 mm EDTA and 0.01 % sodium azide. The flow rate (pump) was ml/h and 14.6-ml fractions were collected. The elution profile is shown in Fig.4. Further treatment of the fractions is described under step 5 b. Step 46: Triton-EDTA Extraction To the combined supernatants from step 3 acetone was added to a final concentration of 90%. Precipitated material was allowed to settle at 4"C, washed two times with one half volume 90% acetone, lyophilized, and extracted two times with 100 ml chloro-

3 I. Hindennach and U. Henning 209 form/methanol (2/1) (dry weight: about 2.5 g). The residue was suspended with 30 ml 0.01 M Tris/HCl, ph 7.5, containing 1.5 % Triton X-100, 5 mm EDTA, and 0.05 /, mercaptoethanol. The suspension was incubated for 30 min at room temperature and centrifuged for 30 min at x g. The pellet was extracted once more with 30ml and a third time with 15ml extraction buffer in the same way. To the combined supernatants acetone was added (final concentration : 90 %) and precipitated material was allowed to settle at 4 C. It was lyophilized after two washes with one half volume 90% acetone (dry weight: about 1 g). The protein composition of the combined supernatants is shown in Fig. 3, that of the residue in Fig. 1. At this step again some protein 11* and small amounts of I are lost. The nature of this protein 11* is also discussed in the Results section. StcJp 56: Chromutography on Biogel P150 (Proteins II*, III, and IV) This can be performed on a column as that used in step 5a. For smaller scale preparations we have used a 4.5 x 110-cm column (gel bed 1.8 1). For such a column 400 mg of material from step 4b dissolved (there is no insoluble material) at 30 C with 20 ml Tris/HCl/sodium dodecylsulfate/edta (see step 5 a) was applied to the column which was run at 30 C with the same buffer as that used in step 5a chromatography. The flow rate was 12 ml/h (by gravity, about 20 cm) and 9.5-ml fractions were collected. The elution profile is shown in Fig. 3. Fractions containing individual proteins were lyophilized, the dry material was dissolved with about 1/10 the original volume of water, and acetone was added to a final concentration of 90 %. Precipitated proteins were washed at least two times with 90% acetone and lyophilized. They were then washed 2 times with distilled water in order to remove residual Tris/HCl, EDTA and water-soluble material that slowly comes off the Biogel [17], and again lyophilized. A completely unmodified protein 11* cannot be recovered this way from the column eluate, see next section for the required procedure. RESULTS AND DISCUSSION Mujor Proteins For the purpose of this paper we shall use a protein terminology adopted before [4,7], and we will compare the isolated proteins with those studied by other workers below and in the following communication. To be isolated were the protein that correspond to the electrophoretically separable bands I (= I a + Ib), II*, 111, and IV. Their respective apparent molecular weights (determined electrophoretically) Fig. 1. Protein composition of material from purficatic~n steps. Electrophoresis method b was used. (A, B), Step 1 envelope fraction, the separation of protein I into bands Ia and Ib can only be seen when smaller amounts of protein (25 pg, A) are applied to the slab gel, and it cannot be observed with electrophoresis method a. (C, D) First and second supernatants, respectively, of the M2 - dodecylsulfate extraction (step 2). (E, F) Residue and supernatant, respectively, of the dodecylsulfate-edta extraction (step 3). (G) Residue of the Triton-EDTA extraction (step 4b). Protein IV is preferentially lost during staining-destaining with this type of electrophoresis and is within the proteins of the starting material for the chromatographic runs as shown in Fig. 3 and 4 4a Trypsin treatment of residue 3 1 Envelope fraction 2 DodecylsuIfate-Mg2+ extraction 3 Dodecylsulfate - EDTA extraction 1 J. J. Triton-EDTA extraction 4b of acetone precipitate of supernatant 3 5a Chromatography of Chromatography of residue 4a supernatant 4b Fig.2. Purification scheme.for proteins I. IP, Ill, and It are , , , and It has been demonstrated before [4] that these proteins belong to the outer cell envelope membrane. Band la and Ib are designated protein I because, as it has now been established (C. J. Schmitges and U. Henning, unpublished work), Ia and Ib are two species of the same polypeptide chain. Fig. 1, 3, and 4 show the electrophoretic profiles of these proteins from E. coli B/r. It is not entirely selfevident where to draw the line between major and minor outer membrane proteins, last not least because it has been shown that the amounts synthesized can vary depending on growth conditions and can differ s. 5b

4 210 Escherichia coli Cell Wall Proteins Fig. 3. Chromatography on Biogel PI50 of step 46 muterial. The electrophoretic profile of the starting materials is shown on the lower right gel. As indicated for gel 1 the sample was (and for gel 2 it was not) boiled before electrophoresis, and the same applies to the two gels of the second peak. The bar below the latter two gels indicates the part of the peak that was collected, the leading edge of this peak contains thc minor bands visible below II* in gel 1. The first peak (void volume) contains very little protein. Chromatography conditions are described in the Methods section. The apparent molecular weights are: 11* 30T, 28000; II* 100 C ; 111, 17000; IV, 9000 Fig.4. Chromatography on Biogel PI50 of srep 4u niateriul. The elcctrophoretic profile of the starting material is shown on the lower right gel. it is somewhat overloaded in order to demonstrate the virtual absence of minor bands. The conditions of chromatography are described in the Methods section. The first peak, representing the void volume, contains very little protein. The apparent molecular weights are: I, 38000; 11, 25000; 111 and IV, see legend Fig.3

5 I. Hindennach and U. Henning 21 1 between different strains [19]. There is no doubt, however, that for proteins1, IT*, and IV the designation is correct since at least for E. coli K12 and B it has been found that between lo5 (I, II*) and about 5 x lo5 (IV) copies of each polypeptide chain can be present per cell [4,5,20,21]. It is less clear if I11 should be called a major protein since it is not yet known just how abundant it is in the outer membrane. We include it because there was reason to suspect [17] that it may be the protein bound to lipopolysaccharide [22], and because it is the only one of the four proteins present in E.coli ghosts that so far had resisted isolation [ 171. An isolation procedure for protein I has been described before [17]. This protein is identical to the matrix protein isolated by Rosenbusch [5J and it can be obtained in pure form also by his method. Another protein (very likely identical to our II*) has been purified by Reithmeier and Bragg [23]. The latter is a heat-modifiable protein that as long as it is not exposed in sodium dodecylsulfate to temperatures above 30 C migrates electrophoretically according to an apparent molecular weight of x Upon boiling in sodium dodecylsulfate presumably due to an unfolding, it exhibits an apparent molecular weight of x 33000, and as far as it has been tested this modification is irreversible. Reithmeier and Bragg s proccdure leads to the purification of the modified protein, a potential disadvantage to structural and immunological studies other than those directed at the primary structure of this protein. Protein IV, the lipoprotein occurs in two forms. One is covalently bound to the murein layer of the cell envelope [20] while the other (about 70% of the total lipoprotein present) is not [21]. The bound form can easily be isolated in pure state [24] since isolation of murein is simple and the protein can be removed from it with lysozyme or trypsin. However, both treatments do not yield unmodified lipoprotein since after lysozyme treatment several repeating murein disaccharide units remain attached to the protein and trypsin leads to the loss of a lysine residue [20]. These facts may be disadvantageous for immunological studies or experiments concerning the reconstitution of the outer membrane. Also, it has so far remained unknown if the free form is identical to the bound form. In addition to the drawbacks mentioned all procedures listed so far do not allow the simultaneous isolation of all proteins in question, a serious financial drawback if the isolation of large quantities of all proteins is desirable. Schnaitman has described the simultaneous purification of several of these proteins [3]. For large-scale preparations, however, there are two disadvantages to this method. His first purification step consists in a DEAE-cellulose chromatography of Triton-EDTA solubilized proteins. We had at that time independently attempted exactly the same separation, and have mentioned before [17] that satisfying separations were not obtained and there were also reproducibility problems. The reason very likely is that the proteins when dissolved in Triton-EDTA form various complexes and certainly are not in solution individually ; chromatography of Triton- EDTA solubilized proteins on Biogel P200 revealed that practically all of them appeared in the void volume. The other disadvantage for a preparative method is that it is rather time-consuming. Also, the heat-modifiable protein is obtained only in its modified form. For all these reasons we have developed a relatively fast procedure that allows the isolation of all proteins in question from one batch of cells and that yields proteins 11* and IV in unmodified forms. The flow sheet of Fig. 2 summarizes the purification procedure which is described in detail in the Methods section. Proteins IF, III, and IV The most difficult problem consisted in the effective and complete separation of proteins I and II*. It was achieved by the Triton-EDTA extraction of the dodecylsulfate-edta extracted residue of step 3 (Fig. 1,3). The Triton-EDTA extract contains no protein I, and about 70 /, of proteins II*, 111, and IV; the separation of the latter three by chromatography on Biogel P150 then was simple (Fig. 3). Protein 11* in the column eluate is present completely in its unmodified form. It cannot, however, be recovered from the fractions by the same procedure that is used for all other proteins (see Methods) without that some (10-20% of the total protein present) modification could occur. This is probably due to the high dodecylsulfate-concentration encountered upon dissolving the lyophilized column fractions. The modification can be completely avoided by precipitating the protein with acetone directly from the eluate and it thus is possible to obtain completely unmodified protein 11* in pure form (Fig. 3). Protein 111 when eluted from the column is heavily contaminated with lipopolysaccharide, and there is also some lipopolysaccharide eluting together with protein IV. In both cases the lipopolysaccharide can be completely removed by extraction with phenol/ chloroform/light petroleum [25]. About 95 of the dry weight of all three proteins can be accounted for by their constituent amino acids (plus the lipid part of protein IV). The amino acid composition of protein IV agreed fairly well with the primary structure [26] of that part of the protein that is covalently bound to murein, and its acid hydrolyzate did not contain muramic acid or glucosamine. The purification procedure does not involve lysozyme or trypsin treatments, thus it yields an unmodified protein IV, i.e.

6 212 Eschericliia coli Cell Wall Proteins that fraction of this protein which is not bound covalently to the murein [21]. Since it has been shown that this free lipoprotein is a precursor of the bound one [21] the purified protein will be analyzed further to establish identity with or differences from the bound form. Protein 1 The residue of step 3 (Fig.4) contains practically all of protein I but was often not entirely free from protein II*, and never free from 111 and some IV. We have shown before [4] that trypsin treatment of a more or less intact outer membrane does not alter proteins I and 111 but splits off part of protein 11* to yield its tryptic fragment protein 11. The two proteins in the step 3 residue exhibit the same behavior, and chromatography on Biogel P150 (Fig. 4) of the material after treatment with trypsin yields protein I in practically quantitative yield. The tryptic fragment of protein II*, when present, can also be obtained (it is well separated from I and 111) and it will be useful for structural studies on this protein. To ascertain that protein I is not altered in a minor way by the action of trypsin we have prepared protein I by Rosenbusch s procedure [5]. The latter preparation yielded precisely the same cyanogen bromide fragments [17] as does protein I after exposure to trypsin. This fact does not yet exclude tryptic removal of a short N-terminal or C-terminal peptide (loss of more than 10 amino acids would easily become detectable by electrophoresis of the cyanogen bromide fragments). Edman-degradation of protein I purified the way described here revealed the N-terminal alanine found also by Rosenbusch [5]. Carboxypeptidase A treatment of protein I purified according to Rosenbusch (no trypsin involved) released the same amino acids as had been found with trypsinized protein I [17]. Therefore, it is practically excluded that the trypsin treatment has any effect on this protein. Concerning all other properties protein I isolated as described here is identical to protein I as we have characterized it before [17]. Yields From 200 g cell paste PZ 120 mg protein I, x 110 mg protein II*, x 50 mg protein 111, and x 30 mg protein IV are obtained. (5-6 days are required for step 1 through steps 4b or 4a, and a complete chromatography takes 5 days.) It may be that the yield of 11* can be increased by a step 2 modification decreasing its loss into the second supernatant (see Methods). The yields of I, II*, and IV are of the expected order of magnitude. Cell counts showed that 200g cell paste consists of 2-3 x loi3 cells. Assuming about lo5 copies per cell each of protein I, II*, and IV (note that the part of protein IV which is covalently bound to murein is not included), quantitative recoveries should yield mg protein I (mol. wt ), mg protein 11* (mol.wt 33000), and mg protein IV (mol. wt 7000). As stated above, the cellular concentration of protein I11 is not known but its yield indicates that this concentration is not an order of magnitude below that of the other proteins. Other Mujor Proteiits For E. coli 0111 Schnaitman has provided evidence that those major outer membrane proteins that are in the molecular weight range consist of (at least) four different polypeptide species [3], and three of these are easily separable electrophoretically. He also reported that one of these proteins (his protein 2) is not normally present in E. coli K12. In confirmation of his results we have never observed a band corresponding to protein 2 in any of our K12 strains nor in the E. coli Bjr used for the purification described here. In addition, he studied a heat-modifiable protein (protein 3, electrophoretic position and heat-modification characteristic as our protein II*) and produced rather strong evidence that it consists of two heat-modifiable polypeptides 3a and 3b. Our purification leads to losses of protein 11* at two steps (see also Methods) : some of it is present in the second supernatant of the dodecylsulfate-mg extraction (step 2, Fig. 1) and some of it is not extractable with Triton-EDTA at step 4b (Fig. 1). At both steps and especially at the latter, therefore, protein with the electrophoretic mobility of protein 11* is discarded and it could well have been that in one or the other case a major protein different from protein 11* (e.g. Schnaitman s protein 3a or 3b) is lost. It has been possible to purify both loss proteins to near electrophoretic homogeneity. The electrophoretic mobility of the cyanogen bromide fragments of both preparations were precisely identical to those of the isolated protein 11* (step 5 b, see the following paper for these fragments). It is thus clear that in the cells we have used there is no major protein other than protein 11* with the same electrophoretic behavior and which would have been lost during purification. It will be shown in the following communication that the same is true for an E. coli K12. The question as to whether or not the isolated 11* is a single polypeptide chain will be taken up in the following communication, and here it suffices to state that the procedure described leads to the isolation of all major proteins. After this manuscript had been completed a paper by Schnaitman et al. [27] appeared showing that this protein 2 occurs as a consequence of lysogenization with a certain phage that obviously is absent from all strains we have in use.

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