100
HETEROLOGOUS EXPRESSION
[10]
[10] H e t e r o l o g o u s E x p r e s s i o n o f M a m m a l i a n P 4 5 0 in C O S Cells
By B A R B A R A
J. CLARK and MICHAEL R. WATERMAN
Introduction The use of heterologous expression systems to study structural and functional aspects of various forms of cytochrome P450 has found wide acceptance in recent years. A primary requirement for such an expression system is that it have low background levels of endogenous P450. As described elsewhere in Section II of this volume, yeast, insect cells, and E s c h e r i c h i a coli meet this requirement. In addition, several different mammalian cell systems have also been found suitable for this purpose, and the transformation and viral infection of such cells are also described elsewhere in Section II. The purpose of this chapter is to describe potential uses and limitations of transient transfection of COS cells to study P450 function and structure. The focus is on transient transfection of COS cells, subcellular fractionation of transfected COS 1 cells, measurement of P450 activity, and quantitation of P450 expression. Transient Expression of Cytochrome P450 cDNA in COS 1 Cells Transformation of African green monkey kidney cells (CV-1) with an origin-defective mutant of simian virus 40 (SV40) viral DNA resulted in the integration of a single copy of the complete early region of SV40 DNA into the CV-1 genome, establishing the COS 1 cell line.l COS cells produce SV40 T antigen which is required for viral replication. Thus, any transiently transfected plasmid DNA which contains the SV40 origin of replication is capable of replicating in COS 1 cells. Transfected cells, therefore, will contain multiple copies of the plasmid which can produce the protein encoded by the eDNA inserted within it. The expression vectors pCD, pSVL, and pCMV have most commonly been used for successful expression of cloned heterologous P450 cDNAs in COS 1 cells. The pCD expression vector contains the SV40 origin of replication and utilizes the SV40 early promoter for transcription of the cloned eDNA. 2 pSVL (Pharmacia, Piscataway, NJ) is similar to pCD but utilizes the SV40 late promoter and contains a multiple cloning site for 1y. Gluzman, Cell (Cambridge, Mass.) 23, 175 (1981). 2H. Okayamaand P. Berg, Mol. Cell. Biol. 3, 280 (1983). METHODS IN ENZYMOLOGY,VOL. 206
Copyright © 1991 by Academic Press, Inc. All rightsof reproductionin any formreserved.
[10]
MAMMALIAN P450 EXPRESSION IN C O S CELLS
101
convenience of cDNA insertion into the expression vector, pCMV is a mammalian expression vector designed for use in cultured cells and contains the promoter regulatory region of the human cytomegalovirus (Towne strain) major immediate early gene upstream of a polylinker cassette for cloning of the cDNA of interest into the vector. 3'4 Compared to the SV40-based vectors, pCMV can lead to approximately a 10-fold greater level of expressed P450 protein in COS 1 c e l l s ) : The COS 1 cell line was first established as a useful system for P450 studies with the expression of the steroidogenic microsomal P450, 17t~hydroxylase. 8 Monooxygenases function in the kidney to metabolize fatty acids, vitamin D, and xenobiotics. The NADPH-cytochrome-P450 reductase, adrenodoxin, and adrenodoxin reductase which are endogenously expressed in COS I cells can functionally interact with transiently expressed cytochrome P450 from several gene families encoding both microsomal and mitochondrial forms of P450. 8-12 In addition, multiple cDNAs can be cotransfected and functionally expressed at the same time to establish a metabolic pathway within these cells. 12 Therefore, this expression system is well suited for functional studies on a wide variety of eukaryotic forms of P450. Methods for Transient Transfection of COS 1 Cells The optimal method for transfection of mammalian cells with plasmid DNA is dependent on the cell type. Calcium phosphate precipitation, DEAE-dextran, electroporation, and lipofection have all been successfully used with COS 1 cells. The details and background of these methods have been well described, and therefore representative protocols used for transfection of P450 cDNA into COS 1 cells are only briefly summarized. 3 D. R. Thomsen, R. M. Stenberg, W. F. Goins, and M. F. Stinski, Proc. Natl. Acad. Sci. U.S.A. 81, 659 (1984). 4 S. Andersson, D. L. Davis, H. Dahlback, H. Jornvall, and D. W. Russell, J. Biol. Chem. 264, 8222 (1989). 5 T. Kronbach, T. M. Larabee, and E. F. Johnson, Proc. Natl. Acad. Sci. U.S.A. 86, 8262 (1989). 6 E. F. Johnson, D. L. Walker, and K. J. Griffin, Biochemistry 29, 873 (1990). 7 B. J. Clark and M. R. Waterman, J. Biol. Chem. 2,66, 5898 (1991). 8 M. X. Zuber, E. R. Simpson, and M. R. Waterman, Science 234, 1258 (1986). 9 C. J. Corbin, S. Graham-Lorence, M. McPhaul, J. I. Mason, C. R. Mendelson, and E. R. Simpson, Proc. Natl. Acad. Sci. U.S.A. 85, 8948 (1988). t0 T. Ichikawa, T. Itakura, and M. Negishi, Biochemistry 28, 4779 (1989). ii C. L. Potenza, M. R. Pendurthi, D. K. Strom, R. H. Tukey, K. J. Griffin, G. E. Schwab, and E. F. Johnson, J. Biol. Chem. 264, 16222 (1989). 12 M. X. Zuber, J. I. Mason, E. R. Simpson, and M. R. Waterman, Proc. Natl. Acad. Sci. U.S.A. 85, 699 (1988).
102
HETEROLOGOUS EXPRESSION
[10]
Plasmid DNA purified on CsCI gradients is suitable for transfection of COS 1 cells.
Calcium Phosphate Precipitation 13-15 Passage COS cells from one confluent 100-mm dish by trypsinization into four 100-mm dishes the day before transfection. Dilute DNA in 140 mM NaCl, 5 mM KC1, 0.75 mM Na2HPO4,6 mM dextrose, 25 mM HEPES, pH 7.1 (HBS), to a concentration of 10-20/zg DNA/ml and slowly add 2 M CaC12 to a final concentration of 125 mM. Incubate the mixture at room temperature for 20-30 min until a visible fine precipitate forms. Remove the medium from the cells and add 1 ml of the precipitate to the cell monolayer. Incubate at room temperature for 15 min, then add 10 ml of fresh medium, and continue incubation at 37° for 4 hr. Replace the medium with HBS plus 15% glycerol (v/v) and let sit for 3 min. 15 Wash cells with fresh medium, then incubate with 8 - l 0 ml of fresh medium (change daily) at 37° until harvest.
DEAE-Dextran 16,17 Passage COS cells from one confluent 100-mm dish by trypsinization into 4 100-mm dishes the day before transfection. Wash COS 1 cells once with Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) buffered with 20 mM HEPES, pH 7.4, plus antibiotics (DM-). Freshly prepare a transfection mix of 2-10/.~g DNA and 5 ~1 of DEAE-dextran (50 mg/ml) per milliliter of D M - medium. Typically one 100-mm dish of COS cells is incubated with 2 ml of DNA/DEAE-dextran transfection mix for 1 hr. The medium is replaced with 8-10 ml of DM plus 10% calf serum (v/v) and antibiotics (DM ÷) with 100/zM chloroquine and incubated for an additional 4-5 hr.~S The medium is removed, and the cells are washed once with DM ÷ medium and incubated at 37° with 8-10 ml of fresh DM ÷ (change daily) until harvest.
Electroporation and Lipofection Plasmid DNA can also be introduced into COS 1 cells by electroporation 19-21or lipofection. 22'23Although there are no reported experiments on the use of either of these methods for P450 studies, the following procedures have been successful. 24 13 F. L. Graham and A. J. van der Eb, Virology 52, 456 (1973). 14 E. Frost and J. Williams, Virology 91, 39 (1978). 15 G. R. Stark and B. A. Parker, J. Virol. 31, 360 (1979). t6 j. H. McCutchan and J. S. Pagano, J. Natl. Cancer Inst. 41, 351 (1968). 17 L. M. Sompaynac and K. J. Danna, Proc. Natl. Acad. Sci. U.S.A. 78, 7575 (1981). Is H. Luthman and G. Magmisson, Nucleic Acids Res. 11, 1295 (1983). t9 E. Neumann, M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider, EMBO J. 1, 841 (1982).
[10]
MAMMALIAN P450 EXPRESSION IN C O S CELLS
103
Electroporation. Collect COS 1 cells from one confluent 100-mm dish by trypsinization. Pellet the cells and resuspend in 1 ml phosphate-buffered saline (PBS) plus 20 mM HEPES, pH 7.4. Add 20/~g plasmid DNA and electroporate at 200-250 V and 1180 f~ capacitance for 1 pulse (CellPorator Electroporation System, Bethesda Research Laboratories, Gaithersburg, MD). Divide the cells into four 100-mm dishes, add 8-10 ml of DM ÷ medium, and incubate at 37° (change medium daily) until harvest. Lipofection. The method of Feigner et al. 22is suggested with the exception that Opti-MEM media (GIBCO) be used for the lipid-DNA incubation with the cultured cells. Comments. Despite the reported improvement in the percentage of cells transformed by electroporation and lipofection techniques, the efficiency of transfection based on P450 activity is comparable between the four methods, with lipofection being about 2 times more effective than the other methods. 24
Subcellular Fractionation of COS 1 Cells Solutions
Phosphate-buffered saline (PBS; Ca2÷,Mg2+-free, GIBCO) Homogenization buffer (HB): 0.5 M sucrose, l0 mM Tris-C1, pH 7.5, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride (PMSF), 0.1/xg/ ml leupeptin, 0.04 units aprotinin/ml Storage buffer: HB except sucrose concentration is 0.25 M Procedure. Harvest cells 72-96 hr posttransfection. 1. Wash the cells with cold PBS and collect the cells from the tissue culture plate in HB by scraping with a rubber policeman. 2. Recover the cell pellet by centrifugation at 800 g for 5 rain (~3000 rpm, Microfuge 12, Beckman, Fullerton, CA). Resuspend the cells in HB to a concentration of 4 x 107 cells/ml HB (one 100-ml confluent tissue culture dish contains - 2 x 106 COS 1 cells). 3. Lyse the cells on ice in a smooth glass tissue grinder (4 ml capacity, Thomas Scientific, Philadelphia, PA) tightly fitted with a Teflon 20 H. Potter, L. Weir, and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 81, 7161 (1984). 21 "Cell-Porator Electroporation System Addendum: Experimental Data." Bethesda Research Laboratories, Gaithersburg, Maryland, 1987. 22 p. L. Feigner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987). 23 A. C. Y. Chang and O. G. Brenner, Focus 10(4), 66 (1988). 24 j. I. Mason, University of Texas Southwestern Medical Center, Dallas, personal communication (1991).
104
HETEROLOGOUSEXPRESSION
4.
5.
6.
7.
[10]
pestle by motor-driven homogenization at 1800 rpm for 25 strokes. Typically COS I cells from ten 100-ram dishes ( - 2 x 10 7 cells) are lysed in 0.5 ml HB; minimally use a five dish (1 x 107 cells) : 0.5 ml HB ratio. Dilute the homogenate with an equal volume of 10 mM Tris-Cl, pH 7.5, 1 mM EDTA and layer over 1/2 volume of HB. Centrifuge at 900 g for 10 min at 4 ° (5000 rpm for 4 min, TLS.55 rotor; Beckman TL100 ultracentrifuge) to remove cell debris and nuclei. The pellet fraction is saved and defined as P1. Remove the supernatant and sucrose pad interface and layer over 1/2 volume HB. Centrifuge 9000 g for l0 min at 4 ° (10,000 rpm, l0 min, TLS.55 rotor; Beckman TL100 ultracentrifuge) to sediment mitochondria. The mitochondrial pellet is defined as P2. Centrifuge the resultant supernatant at 213,000 g for 30 min at 4° (70,000 rpm, TLI00.3 rotor; Beckman TL100 ultracentrifuge) to isolate microsomes. The microsomal pellet is defined as P3, and the final supernatant is the soluble fraction, $3. Resuspend P1, P2, and P3 by hand homogenization in storage buffer.
Comments. The methodology developed for subcellular fractionation of animal tissue in most instances is not directly applicable for cells grown in culture. 25The major difficulty is aggregation of organelles on homogenization, which is believed to be due to an extensive cytoskeletal network in tissue culture c e l l s . 25'26 The salt concentration is critical for fractionation. The above procedure is dependent on endogenous salts of the COS 1 cells; therefore, the cell to buffer volume ratio is an important factor for minimization of variability between experiments. Table I characterizes the subcellular fractions obtained from this procedure. 7 If the yield of recovery of microsomal membranes is crucial, titration of the homogenization buffer to determine empirically the salt concentration required to maximize the recovery should be tried. However, characterization of subcellular fractions by a second method gives a similar marker enzyme distribution to that in Table I. 27 Therefore, with the COS 1 expression system a loss of microsomal membrane recovery owing to organelle aggregation should be expected. 25 R. A. Feldman, S. Gaetani, and T. Morimoto, in "Cancer-Cell Organelles, Methodological Surveys" (E. Reid, G. M. W. Cook, and D. J. Morre, eds.), Vol. 11, p. 263. Ellis Horwood Limited, Chichester, England, 1982. 26 W. W. Franke, D. Mayer, E. Schmid, H. Denk, and E. Borenfreund, Exp. Cell Res. 134, 345 (1981). 27 O. Minowa, K. Sogawa, Y. Higashi, and Y. Fujii-Kuriyama, Cell Struct. Funct. 15, 21 (1990).
0
HETEROLOGOUS EXPRESSION
[10]
[10] H e t e r o l o g o u s E x p r e s s i o n o f M a m m a l i a n P 4 5 0 in C O S Cells
By B A R B A R A
J. CLARK and MICHAEL R. WATERMAN
Introduction The use of heterologous expression systems to study structural and functional aspects of various forms of cytochrome P450 has found wide acceptance in recent years. A primary requirement for such an expression system is that it have low background levels of endogenous P450. As described elsewhere in Section II of this volume, yeast, insect cells, and E s c h e r i c h i a coli meet this requirement. In addition, several different mammalian cell systems have also been found suitable for this purpose, and the transformation and viral infection of such cells are also described elsewhere in Section II. The purpose of this chapter is to describe potential uses and limitations of transient transfection of COS cells to study P450 function and structure. The focus is on transient transfection of COS cells, subcellular fractionation of transfected COS 1 cells, measurement of P450 activity, and quantitation of P450 expression. Transient Expression of Cytochrome P450 cDNA in COS 1 Cells Transformation of African green monkey kidney cells (CV-1) with an origin-defective mutant of simian virus 40 (SV40) viral DNA resulted in the integration of a single copy of the complete early region of SV40 DNA into the CV-1 genome, establishing the COS 1 cell line.l COS cells produce SV40 T antigen which is required for viral replication. Thus, any transiently transfected plasmid DNA which contains the SV40 origin of replication is capable of replicating in COS 1 cells. Transfected cells, therefore, will contain multiple copies of the plasmid which can produce the protein encoded by the eDNA inserted within it. The expression vectors pCD, pSVL, and pCMV have most commonly been used for successful expression of cloned heterologous P450 cDNAs in COS 1 cells. The pCD expression vector contains the SV40 origin of replication and utilizes the SV40 early promoter for transcription of the cloned eDNA. 2 pSVL (Pharmacia, Piscataway, NJ) is similar to pCD but utilizes the SV40 late promoter and contains a multiple cloning site for 1y. Gluzman, Cell (Cambridge, Mass.) 23, 175 (1981). 2H. Okayamaand P. Berg, Mol. Cell. Biol. 3, 280 (1983). METHODS IN ENZYMOLOGY,VOL. 206
Copyright © 1991 by Academic Press, Inc. All rightsof reproductionin any formreserved.
[10]
MAMMALIAN P450 EXPRESSION IN C O S CELLS
101
convenience of cDNA insertion into the expression vector, pCMV is a mammalian expression vector designed for use in cultured cells and contains the promoter regulatory region of the human cytomegalovirus (Towne strain) major immediate early gene upstream of a polylinker cassette for cloning of the cDNA of interest into the vector. 3'4 Compared to the SV40-based vectors, pCMV can lead to approximately a 10-fold greater level of expressed P450 protein in COS 1 c e l l s ) : The COS 1 cell line was first established as a useful system for P450 studies with the expression of the steroidogenic microsomal P450, 17t~hydroxylase. 8 Monooxygenases function in the kidney to metabolize fatty acids, vitamin D, and xenobiotics. The NADPH-cytochrome-P450 reductase, adrenodoxin, and adrenodoxin reductase which are endogenously expressed in COS I cells can functionally interact with transiently expressed cytochrome P450 from several gene families encoding both microsomal and mitochondrial forms of P450. 8-12 In addition, multiple cDNAs can be cotransfected and functionally expressed at the same time to establish a metabolic pathway within these cells. 12 Therefore, this expression system is well suited for functional studies on a wide variety of eukaryotic forms of P450. Methods for Transient Transfection of COS 1 Cells The optimal method for transfection of mammalian cells with plasmid DNA is dependent on the cell type. Calcium phosphate precipitation, DEAE-dextran, electroporation, and lipofection have all been successfully used with COS 1 cells. The details and background of these methods have been well described, and therefore representative protocols used for transfection of P450 cDNA into COS 1 cells are only briefly summarized. 3 D. R. Thomsen, R. M. Stenberg, W. F. Goins, and M. F. Stinski, Proc. Natl. Acad. Sci. U.S.A. 81, 659 (1984). 4 S. Andersson, D. L. Davis, H. Dahlback, H. Jornvall, and D. W. Russell, J. Biol. Chem. 264, 8222 (1989). 5 T. Kronbach, T. M. Larabee, and E. F. Johnson, Proc. Natl. Acad. Sci. U.S.A. 86, 8262 (1989). 6 E. F. Johnson, D. L. Walker, and K. J. Griffin, Biochemistry 29, 873 (1990). 7 B. J. Clark and M. R. Waterman, J. Biol. Chem. 2,66, 5898 (1991). 8 M. X. Zuber, E. R. Simpson, and M. R. Waterman, Science 234, 1258 (1986). 9 C. J. Corbin, S. Graham-Lorence, M. McPhaul, J. I. Mason, C. R. Mendelson, and E. R. Simpson, Proc. Natl. Acad. Sci. U.S.A. 85, 8948 (1988). t0 T. Ichikawa, T. Itakura, and M. Negishi, Biochemistry 28, 4779 (1989). ii C. L. Potenza, M. R. Pendurthi, D. K. Strom, R. H. Tukey, K. J. Griffin, G. E. Schwab, and E. F. Johnson, J. Biol. Chem. 264, 16222 (1989). 12 M. X. Zuber, J. I. Mason, E. R. Simpson, and M. R. Waterman, Proc. Natl. Acad. Sci. U.S.A. 85, 699 (1988).
102
HETEROLOGOUS EXPRESSION
[10]
Plasmid DNA purified on CsCI gradients is suitable for transfection of COS 1 cells.
Calcium Phosphate Precipitation 13-15 Passage COS cells from one confluent 100-mm dish by trypsinization into four 100-mm dishes the day before transfection. Dilute DNA in 140 mM NaCl, 5 mM KC1, 0.75 mM Na2HPO4,6 mM dextrose, 25 mM HEPES, pH 7.1 (HBS), to a concentration of 10-20/zg DNA/ml and slowly add 2 M CaC12 to a final concentration of 125 mM. Incubate the mixture at room temperature for 20-30 min until a visible fine precipitate forms. Remove the medium from the cells and add 1 ml of the precipitate to the cell monolayer. Incubate at room temperature for 15 min, then add 10 ml of fresh medium, and continue incubation at 37° for 4 hr. Replace the medium with HBS plus 15% glycerol (v/v) and let sit for 3 min. 15 Wash cells with fresh medium, then incubate with 8 - l 0 ml of fresh medium (change daily) at 37° until harvest.
DEAE-Dextran 16,17 Passage COS cells from one confluent 100-mm dish by trypsinization into 4 100-mm dishes the day before transfection. Wash COS 1 cells once with Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) buffered with 20 mM HEPES, pH 7.4, plus antibiotics (DM-). Freshly prepare a transfection mix of 2-10/.~g DNA and 5 ~1 of DEAE-dextran (50 mg/ml) per milliliter of D M - medium. Typically one 100-mm dish of COS cells is incubated with 2 ml of DNA/DEAE-dextran transfection mix for 1 hr. The medium is replaced with 8-10 ml of DM plus 10% calf serum (v/v) and antibiotics (DM ÷) with 100/zM chloroquine and incubated for an additional 4-5 hr.~S The medium is removed, and the cells are washed once with DM ÷ medium and incubated at 37° with 8-10 ml of fresh DM ÷ (change daily) until harvest.
Electroporation and Lipofection Plasmid DNA can also be introduced into COS 1 cells by electroporation 19-21or lipofection. 22'23Although there are no reported experiments on the use of either of these methods for P450 studies, the following procedures have been successful. 24 13 F. L. Graham and A. J. van der Eb, Virology 52, 456 (1973). 14 E. Frost and J. Williams, Virology 91, 39 (1978). 15 G. R. Stark and B. A. Parker, J. Virol. 31, 360 (1979). t6 j. H. McCutchan and J. S. Pagano, J. Natl. Cancer Inst. 41, 351 (1968). 17 L. M. Sompaynac and K. J. Danna, Proc. Natl. Acad. Sci. U.S.A. 78, 7575 (1981). Is H. Luthman and G. Magmisson, Nucleic Acids Res. 11, 1295 (1983). t9 E. Neumann, M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider, EMBO J. 1, 841 (1982).
[10]
MAMMALIAN P450 EXPRESSION IN C O S CELLS
103
Electroporation. Collect COS 1 cells from one confluent 100-mm dish by trypsinization. Pellet the cells and resuspend in 1 ml phosphate-buffered saline (PBS) plus 20 mM HEPES, pH 7.4. Add 20/~g plasmid DNA and electroporate at 200-250 V and 1180 f~ capacitance for 1 pulse (CellPorator Electroporation System, Bethesda Research Laboratories, Gaithersburg, MD). Divide the cells into four 100-mm dishes, add 8-10 ml of DM ÷ medium, and incubate at 37° (change medium daily) until harvest. Lipofection. The method of Feigner et al. 22is suggested with the exception that Opti-MEM media (GIBCO) be used for the lipid-DNA incubation with the cultured cells. Comments. Despite the reported improvement in the percentage of cells transformed by electroporation and lipofection techniques, the efficiency of transfection based on P450 activity is comparable between the four methods, with lipofection being about 2 times more effective than the other methods. 24
Subcellular Fractionation of COS 1 Cells Solutions
Phosphate-buffered saline (PBS; Ca2÷,Mg2+-free, GIBCO) Homogenization buffer (HB): 0.5 M sucrose, l0 mM Tris-C1, pH 7.5, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride (PMSF), 0.1/xg/ ml leupeptin, 0.04 units aprotinin/ml Storage buffer: HB except sucrose concentration is 0.25 M Procedure. Harvest cells 72-96 hr posttransfection. 1. Wash the cells with cold PBS and collect the cells from the tissue culture plate in HB by scraping with a rubber policeman. 2. Recover the cell pellet by centrifugation at 800 g for 5 rain (~3000 rpm, Microfuge 12, Beckman, Fullerton, CA). Resuspend the cells in HB to a concentration of 4 x 107 cells/ml HB (one 100-ml confluent tissue culture dish contains - 2 x 106 COS 1 cells). 3. Lyse the cells on ice in a smooth glass tissue grinder (4 ml capacity, Thomas Scientific, Philadelphia, PA) tightly fitted with a Teflon 20 H. Potter, L. Weir, and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 81, 7161 (1984). 21 "Cell-Porator Electroporation System Addendum: Experimental Data." Bethesda Research Laboratories, Gaithersburg, Maryland, 1987. 22 p. L. Feigner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987). 23 A. C. Y. Chang and O. G. Brenner, Focus 10(4), 66 (1988). 24 j. I. Mason, University of Texas Southwestern Medical Center, Dallas, personal communication (1991).
104
HETEROLOGOUSEXPRESSION
4.
5.
6.
7.
[10]
pestle by motor-driven homogenization at 1800 rpm for 25 strokes. Typically COS I cells from ten 100-ram dishes ( - 2 x 10 7 cells) are lysed in 0.5 ml HB; minimally use a five dish (1 x 107 cells) : 0.5 ml HB ratio. Dilute the homogenate with an equal volume of 10 mM Tris-Cl, pH 7.5, 1 mM EDTA and layer over 1/2 volume of HB. Centrifuge at 900 g for 10 min at 4 ° (5000 rpm for 4 min, TLS.55 rotor; Beckman TL100 ultracentrifuge) to remove cell debris and nuclei. The pellet fraction is saved and defined as P1. Remove the supernatant and sucrose pad interface and layer over 1/2 volume HB. Centrifuge 9000 g for l0 min at 4 ° (10,000 rpm, l0 min, TLS.55 rotor; Beckman TL100 ultracentrifuge) to sediment mitochondria. The mitochondrial pellet is defined as P2. Centrifuge the resultant supernatant at 213,000 g for 30 min at 4° (70,000 rpm, TLI00.3 rotor; Beckman TL100 ultracentrifuge) to isolate microsomes. The microsomal pellet is defined as P3, and the final supernatant is the soluble fraction, $3. Resuspend P1, P2, and P3 by hand homogenization in storage buffer.
Comments. The methodology developed for subcellular fractionation of animal tissue in most instances is not directly applicable for cells grown in culture. 25The major difficulty is aggregation of organelles on homogenization, which is believed to be due to an extensive cytoskeletal network in tissue culture c e l l s . 25'26 The salt concentration is critical for fractionation. The above procedure is dependent on endogenous salts of the COS 1 cells; therefore, the cell to buffer volume ratio is an important factor for minimization of variability between experiments. Table I characterizes the subcellular fractions obtained from this procedure. 7 If the yield of recovery of microsomal membranes is crucial, titration of the homogenization buffer to determine empirically the salt concentration required to maximize the recovery should be tried. However, characterization of subcellular fractions by a second method gives a similar marker enzyme distribution to that in Table I. 27 Therefore, with the COS 1 expression system a loss of microsomal membrane recovery owing to organelle aggregation should be expected. 25 R. A. Feldman, S. Gaetani, and T. Morimoto, in "Cancer-Cell Organelles, Methodological Surveys" (E. Reid, G. M. W. Cook, and D. J. Morre, eds.), Vol. 11, p. 263. Ellis Horwood Limited, Chichester, England, 1982. 26 W. W. Franke, D. Mayer, E. Schmid, H. Denk, and E. Borenfreund, Exp. Cell Res. 134, 345 (1981). 27 O. Minowa, K. Sogawa, Y. Higashi, and Y. Fujii-Kuriyama, Cell Struct. Funct. 15, 21 (1990).
0
