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Improved purification of acyl carrier protein
ANALYTICAL
BIOCHEMISTRY
(1980)
102,362-364
Improved CHARLES Department
Purification
of Acyl Carrier
0. ROCK* AND JOHN E. CRONAN,
of Microbiology,
University
of Illinois.
Urbana,
Protein JR. Illinois
61801
Received September 17, 1979 An improved method for the purification of acyl carrier protein from Escherichia coli is described. The method consists of four steps: a 2-propanol extraction, batch adsorption to DEAE-cellulose, ammonium sulfate fractionation, and acid precipitation. The purification can be carried out in a few days and yields between 120 and 150 mg of pure acyl carrier protein per kilogram wet weight of cells.
Acyl carrier protein (ACP)2 is an acidic, asymmetric monomer (1) having a molecular weight of 8847 as determined from the primary sequence (2). ACP plays a central role in lipid biosynthesis in that all the acyl intermediates of fatty acid biosynthesis occur as thioesters of ACP (for review see (3)). The acyl moieties are attached to ACP via the only sulfhydryl group on the protein. This sulfhydryl group is added to ACP posttranslationally by the enzyme holo-ACP synthetase (4) which transfers the 4’-phosphopantetheine prosthetic group of CoA to Ser-36 of ACP. ACP and its thioesters do not possess inherent catalytic activity but serve as substrates for at least a dozen different enzymes in the fatty acid biosynthetic pathway and as acyl donors in phospholipid synthesis (3,5). Therefore, large quantities of ACP and acyl-ACP must be prepared in order to biochemically characterize these enzymes. The previous method for the preparation of ACP consists of six steps, and a yield of 116 mg ACP per kg cells (wet weight) was reported (6). In practice, this yield is seldom achieved with yields of 60 to 80 mg L Present address: Department of Biochemistry, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38101. * Abbreviations: ACP, acyl carrier protein; PIPES, piperazine-N, N’-his (2-ethanesulfonic acid). 0003-2697/80/040362-03$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
362
per kilogram being more common (7,8). The purification scheme (6) also requires the gradient elution of ACP from two DEAE columns and when fractionating large amounts of material, several weeks are needed to load and elute the large columns. In this communication we describe a rapid method for the purification of ACP that circumvents many of these difficulties. ACP sources were frozen Escherichia cofi B cells (Grain Processing) or E. cofi K12 strain UBIOOS grown in a fermenter in a glycerol-tryptone broth. Frozen E. coli cells were thawed overnight at 4°C and the wet weight of the cell paste was determined. The cells were suspended by the addition of 0.1 vol of a stock solution containing 1 M Tris, 1 M glycine, and 0.25 M EDTA, adjusted to pH 8.0 with HCl followed by dilution to a final concentration of 1 g per milliliter with distilled water. This mixture was stirred to make a homogeneous suspension, 30 mg of lysozyme (Sigma) per kilogram was added, and the suspension stirred for 2 h at room temperature. An equal volume of 0.5% Triton X-100 was then added to lyse the cells and the suspension stirred for another hour. The gelatinous mass of DNA extruded by the cells was sheared by a brief homogenization (1-2 min) in a Waring blender. To this suspension an equal volume of 2-propanol is slowly
IMPROVED
PURIFICATION
OF ACYL CARRIER PROTEIN
363
minimal volume (ca., 20 mg/ml) and the pH titrated to 7.0 to 7.5 with Tris base. Proteins contaminating the ACP preparation at this stage were removed by the addition of 4 vol of saturated ammonium sulfate. The precipitate was allowed to flocculate for 1 h and the suspension centrifuged at 8OOOg for 20 min to remove the precipitate. ACP was recovered from the supernatant by titrating the pH to 3.9 and collecting the ACP precipitate by centrifugation as described above. Protein concentration was determined by the method of Murphy and Kies (9). SDS or SDS-urea gel electrophoresis (1) of ACP prepared by this method showed only a single band corresponding to ACP. The ACP preparations have also been W*v?xENGTH , nm demonstrated to be pure as judged by FIG. 1. Ultraviolet spectrum of ACP. The spectra amino acid analysis, gel filtration chromawas obtained on an ACP sample in IO mM PIPES, tography, isoelectric focusing, and activity pH 6.8, at a concentration of 2 m&ml using a Cary in the acyl-ACP synthetase reaction. HowModel 14 recording spectrophotometer. ever, the excess absorbance at 260 nm noted in other ACP preparations (1) is usually still added with continual stirring. The mixture is present. This excess absorbance most likely allowed to stand with intermittent stirring for arises from the coelution of nucleic acid 1 h and then centrifuged at 40008 for 20 min components with ACP from the DEAEand the 2-propanol supernatant saved. The cellulose column. This excess absorbance 2-propanol supematant is titrated to pH 6.1 can be removed by repeated acid prewith acetic acid followed by the addition of cipitation at pH 3.9 as described above. The ultraviolet spectrum of pure ACP is 100 ml of defined DEAE- cellulose (DE-23, Whatman) per kilogram of cells and the shown in Fig. 1. ACP exhibits an absorpsuspension stirred overnight to adsorb the tion maximum at 276 nm and a molar ACP to the DE-23. The DE-23 was then extinction at 280 nm of 1.8 x 103. ACP concollected on a large filter funnel and washed tains one tyrosine and two phenylalanines with 5 vol of 10 mM PIPES, pH 6.1, 0.25 M and the spectrum is close to that calculated LiCl, 0.1% Tritonfollowed by 5 vol of 10 mM from this composition. The major abPIPES, pH 6.1,0.25 M LiCl. The DE23 was sorbance is due to the tyrosine and the then recovered from the funnel and packed roughness of the spectrum between 250 and into a column. The ACP fraction was eluted 265 nm (10) is due to the contribution of from the column with 10 mM PIPES, pH 6.1, the phenylalanine residues. containing 0.5 M LiCl. The fractions conThis procedure results in ACP yields of taining ACP are characteristically dark 120 to 150 mg/kg (wet weight) of cells. The brown. These fractions were pooled and the major loss of ACP occurred during the 2pH titrated to 3.9 with acetic acid and the propanol extraction. If the cells were initially mixture allowed to stand overnight to suspended in a smaller volume of buffer completely precipitate the protein. The ACP than called for in the described method the was recovered by centrifugation at SOOOg losses of ACP were found to be greater. for 20 min and the pellet resuspended in a This result was most probably due to the
364
ROCK AND CRONAN
trapping of ACP in the voluminous precipitate that was formed after the addition of 2-propanol. Resuspension and washing of the 2-propanol precipitate was found to be difficult due to its pasty consistency. Therefore, the volume reported in this paper was a compromise between high yields of ACP following 2-propanol extraction and manageable volumes for the subsequent centrifugation steps. This notion was further supported by our experiments on small cultures of E. co/i cells labeled with D[2-3H]pantothenic acid. In these experiments recoveries of [3H]ACP have been found to be routinely greater than 90%. This procedure for the isolation of ACP offers several advantages over the previous method (6). First, we have taken advantage of the high solubility of ACP in 2-propanol to design a first step that removes 95% of the protein from the sample. This step dispenses with the need for breaking cells in a French press, and the streptomycin sulfate and ammonium sulfate fractionation steps in the previous method (6). Second, the time required to complete the purification has been reduced from several weeks to several days. The major time savings result from the elimination of the two large ion-exchange columns called for in the previous method. Finally, our yield of 120 to 150 mg/kg cells is considerably better
than the 60 to 80 mg/kg cells usually obtained using the previous method (7,8). This procedure is also applicable to the extraction of small quantities of labeled ACP from cells in metabolic experiments. ACKNOWLEDGMENT This work was supported by the National Institutes of Allergy and Infectious Diseases Grant AI 1056.
REFERENCES
2. 3. 4. 5. 6.
7.
8. 9. 10.
Rock, C. O., and Cronan, J. E., Jr. (1979) J. Bid. Chem. 254, 9778-9785. Vanaman, T. C., Wakil, S. J., and Hill, R. L. (1968) J. Biol. Chem. 243, 6420-643 1. Vageios, P. R. (1971) Curr. Top. Cell. Reg. 4, 119166. Elovson, J., and Vagelos, P. R. (1968) J. Biol. Chem. 243, 3603-3611. Ray, T. K., and Cronan, J. E., Jr. (1975) J. Biol. Chem. 250, 8422-8427. Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1969) In Methods in Enzymology (J. M. Lowenstein, ed.), Vol. 14, pp. 43-50, Academic Press, New York. Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1%8)In Biochemical Preparations (Lands, W. E. M., ed.), Vol. 12, pp. 56-65, Wiley, New York. Spencer, A. K., Greenspan, A. D., and Cronan, J. E. Jr. (1979) FEB.9 Let?. 101, 253-265. Murphy, J. B., and Kies, M. W. (1960) Biochim. Biophys. Acta 45, 382-384. Pugh, E. L., and Wakil, S. J. (1965) J. Biol. Chem. 240, 4727-4733.
BIOCHEMISTRY
(1980)
102,362-364
Improved CHARLES Department
Purification
of Acyl Carrier
0. ROCK* AND JOHN E. CRONAN,
of Microbiology,
University
of Illinois.
Urbana,
Protein JR. Illinois
61801
Received September 17, 1979 An improved method for the purification of acyl carrier protein from Escherichia coli is described. The method consists of four steps: a 2-propanol extraction, batch adsorption to DEAE-cellulose, ammonium sulfate fractionation, and acid precipitation. The purification can be carried out in a few days and yields between 120 and 150 mg of pure acyl carrier protein per kilogram wet weight of cells.
Acyl carrier protein (ACP)2 is an acidic, asymmetric monomer (1) having a molecular weight of 8847 as determined from the primary sequence (2). ACP plays a central role in lipid biosynthesis in that all the acyl intermediates of fatty acid biosynthesis occur as thioesters of ACP (for review see (3)). The acyl moieties are attached to ACP via the only sulfhydryl group on the protein. This sulfhydryl group is added to ACP posttranslationally by the enzyme holo-ACP synthetase (4) which transfers the 4’-phosphopantetheine prosthetic group of CoA to Ser-36 of ACP. ACP and its thioesters do not possess inherent catalytic activity but serve as substrates for at least a dozen different enzymes in the fatty acid biosynthetic pathway and as acyl donors in phospholipid synthesis (3,5). Therefore, large quantities of ACP and acyl-ACP must be prepared in order to biochemically characterize these enzymes. The previous method for the preparation of ACP consists of six steps, and a yield of 116 mg ACP per kg cells (wet weight) was reported (6). In practice, this yield is seldom achieved with yields of 60 to 80 mg L Present address: Department of Biochemistry, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38101. * Abbreviations: ACP, acyl carrier protein; PIPES, piperazine-N, N’-his (2-ethanesulfonic acid). 0003-2697/80/040362-03$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
362
per kilogram being more common (7,8). The purification scheme (6) also requires the gradient elution of ACP from two DEAE columns and when fractionating large amounts of material, several weeks are needed to load and elute the large columns. In this communication we describe a rapid method for the purification of ACP that circumvents many of these difficulties. ACP sources were frozen Escherichia cofi B cells (Grain Processing) or E. cofi K12 strain UBIOOS grown in a fermenter in a glycerol-tryptone broth. Frozen E. coli cells were thawed overnight at 4°C and the wet weight of the cell paste was determined. The cells were suspended by the addition of 0.1 vol of a stock solution containing 1 M Tris, 1 M glycine, and 0.25 M EDTA, adjusted to pH 8.0 with HCl followed by dilution to a final concentration of 1 g per milliliter with distilled water. This mixture was stirred to make a homogeneous suspension, 30 mg of lysozyme (Sigma) per kilogram was added, and the suspension stirred for 2 h at room temperature. An equal volume of 0.5% Triton X-100 was then added to lyse the cells and the suspension stirred for another hour. The gelatinous mass of DNA extruded by the cells was sheared by a brief homogenization (1-2 min) in a Waring blender. To this suspension an equal volume of 2-propanol is slowly
IMPROVED
PURIFICATION
OF ACYL CARRIER PROTEIN
363
minimal volume (ca., 20 mg/ml) and the pH titrated to 7.0 to 7.5 with Tris base. Proteins contaminating the ACP preparation at this stage were removed by the addition of 4 vol of saturated ammonium sulfate. The precipitate was allowed to flocculate for 1 h and the suspension centrifuged at 8OOOg for 20 min to remove the precipitate. ACP was recovered from the supernatant by titrating the pH to 3.9 and collecting the ACP precipitate by centrifugation as described above. Protein concentration was determined by the method of Murphy and Kies (9). SDS or SDS-urea gel electrophoresis (1) of ACP prepared by this method showed only a single band corresponding to ACP. The ACP preparations have also been W*v?xENGTH , nm demonstrated to be pure as judged by FIG. 1. Ultraviolet spectrum of ACP. The spectra amino acid analysis, gel filtration chromawas obtained on an ACP sample in IO mM PIPES, tography, isoelectric focusing, and activity pH 6.8, at a concentration of 2 m&ml using a Cary in the acyl-ACP synthetase reaction. HowModel 14 recording spectrophotometer. ever, the excess absorbance at 260 nm noted in other ACP preparations (1) is usually still added with continual stirring. The mixture is present. This excess absorbance most likely allowed to stand with intermittent stirring for arises from the coelution of nucleic acid 1 h and then centrifuged at 40008 for 20 min components with ACP from the DEAEand the 2-propanol supernatant saved. The cellulose column. This excess absorbance 2-propanol supematant is titrated to pH 6.1 can be removed by repeated acid prewith acetic acid followed by the addition of cipitation at pH 3.9 as described above. The ultraviolet spectrum of pure ACP is 100 ml of defined DEAE- cellulose (DE-23, Whatman) per kilogram of cells and the shown in Fig. 1. ACP exhibits an absorpsuspension stirred overnight to adsorb the tion maximum at 276 nm and a molar ACP to the DE-23. The DE-23 was then extinction at 280 nm of 1.8 x 103. ACP concollected on a large filter funnel and washed tains one tyrosine and two phenylalanines with 5 vol of 10 mM PIPES, pH 6.1, 0.25 M and the spectrum is close to that calculated LiCl, 0.1% Tritonfollowed by 5 vol of 10 mM from this composition. The major abPIPES, pH 6.1,0.25 M LiCl. The DE23 was sorbance is due to the tyrosine and the then recovered from the funnel and packed roughness of the spectrum between 250 and into a column. The ACP fraction was eluted 265 nm (10) is due to the contribution of from the column with 10 mM PIPES, pH 6.1, the phenylalanine residues. containing 0.5 M LiCl. The fractions conThis procedure results in ACP yields of taining ACP are characteristically dark 120 to 150 mg/kg (wet weight) of cells. The brown. These fractions were pooled and the major loss of ACP occurred during the 2pH titrated to 3.9 with acetic acid and the propanol extraction. If the cells were initially mixture allowed to stand overnight to suspended in a smaller volume of buffer completely precipitate the protein. The ACP than called for in the described method the was recovered by centrifugation at SOOOg losses of ACP were found to be greater. for 20 min and the pellet resuspended in a This result was most probably due to the
364
ROCK AND CRONAN
trapping of ACP in the voluminous precipitate that was formed after the addition of 2-propanol. Resuspension and washing of the 2-propanol precipitate was found to be difficult due to its pasty consistency. Therefore, the volume reported in this paper was a compromise between high yields of ACP following 2-propanol extraction and manageable volumes for the subsequent centrifugation steps. This notion was further supported by our experiments on small cultures of E. co/i cells labeled with D[2-3H]pantothenic acid. In these experiments recoveries of [3H]ACP have been found to be routinely greater than 90%. This procedure for the isolation of ACP offers several advantages over the previous method (6). First, we have taken advantage of the high solubility of ACP in 2-propanol to design a first step that removes 95% of the protein from the sample. This step dispenses with the need for breaking cells in a French press, and the streptomycin sulfate and ammonium sulfate fractionation steps in the previous method (6). Second, the time required to complete the purification has been reduced from several weeks to several days. The major time savings result from the elimination of the two large ion-exchange columns called for in the previous method. Finally, our yield of 120 to 150 mg/kg cells is considerably better
than the 60 to 80 mg/kg cells usually obtained using the previous method (7,8). This procedure is also applicable to the extraction of small quantities of labeled ACP from cells in metabolic experiments. ACKNOWLEDGMENT This work was supported by the National Institutes of Allergy and Infectious Diseases Grant AI 1056.
REFERENCES
2. 3. 4. 5. 6.
7.
8. 9. 10.
Rock, C. O., and Cronan, J. E., Jr. (1979) J. Bid. Chem. 254, 9778-9785. Vanaman, T. C., Wakil, S. J., and Hill, R. L. (1968) J. Biol. Chem. 243, 6420-643 1. Vageios, P. R. (1971) Curr. Top. Cell. Reg. 4, 119166. Elovson, J., and Vagelos, P. R. (1968) J. Biol. Chem. 243, 3603-3611. Ray, T. K., and Cronan, J. E., Jr. (1975) J. Biol. Chem. 250, 8422-8427. Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1969) In Methods in Enzymology (J. M. Lowenstein, ed.), Vol. 14, pp. 43-50, Academic Press, New York. Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1%8)In Biochemical Preparations (Lands, W. E. M., ed.), Vol. 12, pp. 56-65, Wiley, New York. Spencer, A. K., Greenspan, A. D., and Cronan, J. E. Jr. (1979) FEB.9 Let?. 101, 253-265. Murphy, J. B., and Kies, M. W. (1960) Biochim. Biophys. Acta 45, 382-384. Pugh, E. L., and Wakil, S. J. (1965) J. Biol. Chem. 240, 4727-4733.