506
ENZYMOLOGY AND METABOLISM
[58]
2
°~" -
,9.0 E E
1-t
o o t!
!
"7, o
0
E e-
I
I
2
4
6
all-trans--retinol (I~M)
FIG. 1. Dependence of aU-trans-~ll-cis-retinol isomerization rate on all-trans-retinol concentration. The assays were carried out by incubating 0.30 mg of CHAPS-solubilized protein from nuclear membrane in the presence of 81/zM BSA and variable concentrations of all-trans-retinol in a total volume of 1.1 ml. Data from double reciprocal plot (inset) were used to calculate the apparent Vmaxand Km values for the reaction. described, the rate of 11-cis-retinol formation a p p r o a c h e s saturation at about 5 tzM all-trans-retinol. T h e linear transformation of the saturation c u r v e gives an a p p a r e n t Vmax o f 2.5 n m o l / h r / m g protein and a Km of 1.6 /xM (Fig. 1).
[58] Structure-Function Analyses of Mammalian Cellular Retinol-Binding Proteins by Expression in Escherichia coli By MARC S. LEVIN, ELLEN LI, and JEFFREY I. GORDON Rationale for Prokaryotic Expression of Cellular Retinoid-Binding Proteins Several small ( - 1 5 kDa) intracellular retinoid-binding proteins have b e e n purified f r o m animal tissues and their p r i m a r y structures deterMETHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[58]
RAT C R B P AND C R B P II EXPRESSED IN E. coli
507
mined. These include cellular retinol-binding protein (CRBP), 1-3 cellular retinol-binding protein II (CRBP I I ) , 4'5 and cellular retinoic acid-binding protein. 6 They belong to a family of homologous, cytoplasmic, hydrophobic ligand-binding proteins which currently contains 10 known members, several of which bind long-chain fatty acids (reviewed by Sweetser et al.7).
Attempts to rapidly and easily purify these cellular retinoid binding proteins from their cells of origin have been limited by a number of factors. First, the yields of purified protein have been poor: 15/xg of CRBP per gram wet weight of rat liver and 170/zg of CRBP II per gram wet weight of rat small intestine. 8,9 Second, copurification of comparably sized, homologous cellular retinoid-binding proteins may occur when several are expressed in a given tissue. Last, the purified proteins contain bound ligand. To analyze apoprotein-ligand interactions, UV irradiation or organic extraction is required to remove endogenous retinoids, methods which may perhaps lead to structural modifications that could complicate interpretation of subsequent functional studies. By contrast, purification of these mammalian proteins after their expression in Escherichia coli offers several advantages: (1) large quantities of protein are easily obtained; (2) only one retinoid-binding protein is expressed because E. coli does not possess any endogenous retinoid-binding proteins; (3) because E. coli does not require retinoids for growth and endogenous retinoids are not present, the retinoid-binding proteins purified from this prokaryote can be obtained as apoproteins; (4) site-directed mutagenesis of cloned cDNAs encoding retinoid-binding proteins and their subsequent expression in E. coli represent a powerful method for analyzing protein structure-activity relationships; and (5) amino acid derivatives can be introduced into the proteins by growing suitable E. coli auxotrophs containing a prokaryotic expression vector in the presence of the analog. Analog-substituted retinoid-binding proteins can, in turn, be used for nui M. M. Bashor, D. O. Tort, and F. Chytil, Proc. Natl. Acad. Sci. U.S.A. 70, 3483 (1973). 2 D. E. Ong and F. Chytil, J. Biol. Chem. 253, 828 (1978). 3 j. Sundelin, H. Anundi, L. Tragardh, U. Erikson, P. Lind, H. Ronne, P. A, Peterson, and L. Rusk, J. Biol. Chem. 260, 6488 (1985). 4 D. E. Ong, J. Biol. Chem. 259, 1476 (1984). 5 E. Li, L. A. Detainer, D. A. Sweetser, D. E. Ong, and J. I. Gordon, Proc. Natl. Acad. Sci. U.S.A. 83, 5779 (1986). 6 j. Sundelin, S. R. Das, U. Eriksson, L. Rask, and P. A. Peterson, J. Biol. Chem. 260, 6495 (1985). 7 D. A. Sweetser, R. O. Heuckeroth, and J. I. Gordon, Annu. Reo. Nutr. 7, 337 (1987). 8 D. E. Ong, A. J. Crow, and F. Chytii, J. Biol. Chem. 257, 13385 (1982). 9 W. H. Schaefer, B. Kakkad, J. A. Crow, I. A. Blair, and D. E. Ong, J. Biol. Chem. 264, 4212 (1989).
508
ENZYMOLOGY AND METABOLISM
[58]
clear magnetic resonance (NMR) studies of ligand-protein interactions (see below). Expression of Cellular Retinol-Binding Proteins in Escherichia coli General Principles For a general discussion of useful strategies for expressing, identifying, and characterizing recombinant gene products in E. coli, the reader is referred to the review by Shatzman and Rosenberg in this series.l° Two desirable components of prokaryotic expression vectors are (I) inducible promoters which can direct efficient transcription of foreign cDNAs and (2) translational control elements that allow efficient initiation of translation of the foreign mRNA transcript to occur. We have used pMON vectors 11 designed and provided by Monsanto z but a large number of commercially available expression vectors are also suitable. 12The pMON series of vectors are derived from pBR32713 and incorporate the E. coli recA promoter which can be induced by nalidixic acid and a ribosome binding site from the T7 bacteriophage gene 10 leader (G10L) sequence. II The pMON vectors that we have used contain a unique NdeI restriction site downstream from the recA promoter and the translational control element. By genetically engineering an NdeI site at its initiator ATG codon (if necessary), the cDNA of interest can be placed under the control of the recA promoter. Moreover, the distance between the vectorderived G10L ribosome-binding site and its translation start site is thereby maintained. One of the advantages of such a vector is that it directs the synthesis of a full-length "recombinant" polypeptide rather than one which represents a fusion between bacterial and eukaryotic protein sequences. The bacterial strain used for expression of the recombinant protein must be selected with several caveats in mind. First, the induction system used must be compatible with the bacterial phenotype. For example, a recA- strain cannot be used if the E. coli recA promoter is utilized. Second, E. coli proteases such as the product of the lon gene (protease La) may cause proteolysis of some foreign proteins. Expression of these proteins may be improved by using strains that are protease deficient (e.g., Lon- and Htpr-). Third, variables that can affect the efficiency of ~0A. R. Shatzman and M. Rosenberg, this series, Vol. 152, p. 661. H p. O. Olins, C. S. Devine, S. H. Rangwala, and K. S. Kavka, Gene 73, 227 (1988). lz Examples of currently available vectors that are suitable for production of "nonfusion" proteins in E. coli include pBTac2 and pBTrp2 (Boehringer Mannheim Biochemicals); pNHI8A (Stratagene); and pKK233-2 and pPL Lambda (Pharmacia LKB Biotechnology). 13 X. Sober6n, L. Covarrubias, and F. Bolivar, Gene 9, 287 (1980).
[58]
RAT CRBP AND CRBP II EXPRESSEDIN E. coli
509
production of recombinant proteins include incubation temperature, growth medium, cell density, timing of induction, and the length of fermentation after induction of foreign protein synthesis. Finally, it should also be noted that E. coli cannot support many critical posttranslational modifications of mammalian proteins (e.g., glycosylation). Growth o f Escherichia coli Transformed with p M O N - C R B P or pMON-CRBP H
We have not found it necessary to utilize protease-deficient hosts for the expression of CRBP and CRBP II. For most purposes, we use E. coli strain JM101 transformed with pMON-based vectors (Monsanto). A fresh overnight culture~ of E. coli JM101 containing the recombinant pMON vector is diluted 1 : 50 in fresh Luria broth plus ampicillin (100 /~g/ml) (pMON contains an A m p r locus). After achieving a cell density equivalent to an OD600 of approximately 0.2 nm, nalidixic acid is added (1:200 dilution of a 10 mg/ml stock prepared in 0.1 N NaOH) to activate the recA promoter of the plasmid. Fermentation is continued with agitation for an additional 3 hr at 30° (at which time the OD60o is -1.0). Cells are then harvested by centrifugation at 5,000 g or by filtration through a Millipore (Bedford, MA) Pellicon cassette (the Pellicon cassette is useful for harvesting cells from large-scale cultures, i.e., 20-100 liters). The cell paste is stored at - 7 0 °. Purification o f Escherichia coli Expressed Rat CRBP and CRBP H
Although foreign proteins that are expressed in E. coli may be soluble, many are present as insoluble aggregates known as inclusion bodies (reviewed by Marstonl4). Recovery of active proteins from these aggregates requires isolation of the inclusion bodies following cell lysis, subsequent denaturation to solubilize the protein, and finally a refolding reaction. Because soluble proteins can be purified directly, they are more likely to retain their native conformation. At the conclusion of the fermentation described in the preceding section, rat CRBP or CRBP II represents 1015% of the total soluble proteins present in E. coli homogenates. The purification of rat apo CRBP and apo CRBP II from E. coli is remarkably straightforward and relatively rapid, requiring two or t h r e e steps. 15,16Our protocol is summarized in Table I. An aliquot of the E. coli i4 A. Marston, Biochem. J. 240, 1 (1986). 15 E. Li, B. Locke, N. C. Yang, D. E. Ong, and J. I. Gordon, J. Biol. Chem. 262, 13773 (1987). t6 M. S. Levin, B. Locke, N. C. Yang, E. Li, and J. I. Gordon, J. Biol. Chem. 263, 17715 (1988).
510
ENZYMOLOGY AND METABOLISM
[58]
TABLE I PURIFICATIONOF RAT APo-CRBP AND APo-CRBP II FROMEscherichia coli LYSATES 1. 2. 3. 4.
5. 6. 7. 8. 9.
Thaw cell pellet and resuspend in 2-3 volumes cold lysis buffer (see text). Disrupt bacteria with a French press (-2000 psi). Clear lysate by centrifugation at 5000 g for 10 min (4°). Ammonium sulfate fractionation: Drip in an equal volume of cold filtered 100% saturated ammonium sulfate (Whatman) containing 15 mM dithiothreitol (pH adjusted to 7.1 with KOH) over 4 hr (keep on ice). Centrifuge as above to obtain a 50% ammonium sulfate supernatant. For CRBP II only, bring the 50% supernatant to 70% saturation by the addition of solid ammonium sulfate. Centrifuge as above to obtain the 50-70% pellet. Suspend this pellet in lysis buffer. Dialyze the ammonium sulfate fractions (-0.1-0.2 liters) overnight at 4° against -20 liters potassium phosphate (20 mM, pH 7.1), EDTA (1 mM), 2-mercaptoethanol (10 mM), sodium azide (0.05%), phenylmethylsulfonyl fluoride (0.02%, pH 7.1). Concentrate the postdialysis preparations, if necessary, to afinal protein concentration of - 5 mg/ml with an Amicon YMI0 membrane. Perform gel-filtration column chromatography with Sephadex G-50. Screen fractions by absorbance at 280 nm and by SDS-PAGE. Pool fractions containing CRBP or CRBP II. If the acyl cartier protein of E. coli is present (detected by SDS-PAGE and aminoterminal amino acid sequencing) or if separation of Met + from Met- CRBP is desired, then proceed to FPLC, Step 9. Perform FPLC with a Mono Q (Pharmacia) column equilibrated with imidazole (20 mM, pH 6.8) and eluted with a NaC1 gradient (see Fig. IB for details).
cell paste is first thawed to r o o m t e m p e r a t u r e and resuspended in 2 - 3 volumes of lysis buffer [Tris (final concentration 50 m M , p H 7.9), sucrose (10%), and p h e n y l m e t h a n e s u l f o n y l fluoride (0.5 mM)]. Bacteria are disrupted b y p a s s a g e through a F r e n c h Press ( - 2 0 0 0 psi). Cellular debris is r e m o v e d b y centrifugation at 5000 g for 10 min at 4 °. A m m o n i u m sulfate fractionation of the supernatant fraction is a v e r y useful first step in the purification: m o s t bacterial proteins are insoluble in solutions of NH4SO4 at 50% saturation, in contrast to the cellular retinol-binding proteins which remain soluble. Thus, the supernatant is adjusted slowly to 50% saturation with NH4SO4 (with constant stirring at 0-4°). After gentle stirring for an additional hour, the suspension is subjected to centrifugation at 41,000 g for 30 min at 4 °. An additional 70% NH4SO4 " c u t " is also beneficial w h e n purifying C R B P II. The o v e r 50% NH4SO4 supernatant containing C R B P or the 50-70% NH4SO4 precipitate containing C R B P II is then dialyzed overnight at 4 ° against buffer A [potassium p h o s p h a t e (20 m M , p H 7.1), E D T A (1 m M ) , glycerol (15%), sodium azide (0.05%), phenylmethanesulfonyl fluoride (0.5 m M ) , and 2 - m e r c a p t o e t h a n o l (10 mM)]. The dialyzed protein solution is then fractionated through a S e p h a d e x G-50-80 column equilibrated with the s a m e buffer. C o l u m n fractions are a s s a y e d for protein b y mea-
[58]
RAT C R B P AND C R B P II EXPRESSED IN E. coil
511
suring the absorbance at 280 nm. Selected fractions are then surveyed by electrophoresis through a 15% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS-PAGE). Typically, CRBP and CRBP II elute from the gel-filtration column at a relative retention volume (Ve/Vo) of 1.5 (see Fig. 1A). When purifying CRBP (but not CRBP II) an additional purification step is desirable to avoid contamination with the acyl carrier protein of E. coli. The acyl carrier protein is a soluble 8.8-kDa protein which migrates on SDS-PAGE as an approximately 20-kDa protein and elutes like a 12-kDa protein from Sephadex G-50 (reviewed by Rock and Cronan17). Those fractions which appear to be enriched for CRBP by SDS-PAGE are pooled and further purified by fast protein liquid chromatography (FPLC). The pooled fractions are applied to a Pharmacia (Piscataway, NJ) Mono Q column equilibrated with imidazole (20 mM, pH 6.8) and sodium azide (0.05%). CRBP is eluted with a continuous NaCI gradient (15.5-25 mM) in the same buffer. Two CRBP peaks elute at approximately 19 and 20 m M NaC1 (see Fig. 1B). These peaks correspond to CRBP without and with its initiator methionine residue, respectively. The proportion of Met + CRBP and Met- CRBP varies from protein preparation to preparation. The initiator methionine of foreign proteins expressed in E. coli may be removed coincidently, or shortly after synthesis, by a nonspecific aminopeptidase. ~8,19 The efficiency of methionine removal from recombinant proteins is variable and affected by the physicochemical properties of the penultimate amino acid. 18,~9The spectrofluorimetric and ligand binding properties of Met + and Met- CRBP appear to be identical. 16 For some applications, such as protein crystallization and NMR studies, it may be desirable to separate the two species (see below). Protein integrity and purity can be assessed by SDS-PAGE, isoelectric focusing, and automated sequential Edman degradation. Unlike the forms recovered from mammalian cells or cell-free translation systems (wheat germ or reticulocyte lysates), E. coli-derived rat CRBP and CRBP II do not have an acetyl group linked to their amino-terminal amino acid. 15,16Therefore, the intact E. coli-derived proteins can be sequenced directly. Purified E. coli-derived rat apo-CRBP and apo-CRBP II are stored in plastic tubes at 4° in phosphate buffer (20 mM, pH 7.4) supplemented with 2-mercaptoethanol (1 mM), EDTA (1 mM), and NaN3 (0.05%). We have not found aggregation to be a problem even at protein concentrations as high as 15 mg/ml ( - 1 mM). 17 C. O. Rock and J. E. Cronan, Jr., this series, Vol. 71, p. 341. 18 j. L. Brown, Biochim. Biophys. Acta 221, 480 (1970). 19j. L. Brown and J. F. Krall, Biochem. Biophys. Res. Commun. 42, 390 (1971).
512
ENZYMOLOGY AND METABOLISM
[58]
A vo 66453629-
?
_o 24-
c
I.C
' 20.1-
o co oq >,, 4J
14.2 -
g 0
~0.50
Ve
/ 1.00
1.50 Elution Volume
2.00
(liters)
B E
c 0
500 400
O~ O C t~ 0 .Q ,
ENZYMOLOGY AND METABOLISM
[58]
2
°~" -
,9.0 E E
1-t
o o t!
!
"7, o
0
E e-
I
I
2
4
6
all-trans--retinol (I~M)
FIG. 1. Dependence of aU-trans-~ll-cis-retinol isomerization rate on all-trans-retinol concentration. The assays were carried out by incubating 0.30 mg of CHAPS-solubilized protein from nuclear membrane in the presence of 81/zM BSA and variable concentrations of all-trans-retinol in a total volume of 1.1 ml. Data from double reciprocal plot (inset) were used to calculate the apparent Vmaxand Km values for the reaction. described, the rate of 11-cis-retinol formation a p p r o a c h e s saturation at about 5 tzM all-trans-retinol. T h e linear transformation of the saturation c u r v e gives an a p p a r e n t Vmax o f 2.5 n m o l / h r / m g protein and a Km of 1.6 /xM (Fig. 1).
[58] Structure-Function Analyses of Mammalian Cellular Retinol-Binding Proteins by Expression in Escherichia coli By MARC S. LEVIN, ELLEN LI, and JEFFREY I. GORDON Rationale for Prokaryotic Expression of Cellular Retinoid-Binding Proteins Several small ( - 1 5 kDa) intracellular retinoid-binding proteins have b e e n purified f r o m animal tissues and their p r i m a r y structures deterMETHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[58]
RAT C R B P AND C R B P II EXPRESSED IN E. coli
507
mined. These include cellular retinol-binding protein (CRBP), 1-3 cellular retinol-binding protein II (CRBP I I ) , 4'5 and cellular retinoic acid-binding protein. 6 They belong to a family of homologous, cytoplasmic, hydrophobic ligand-binding proteins which currently contains 10 known members, several of which bind long-chain fatty acids (reviewed by Sweetser et al.7).
Attempts to rapidly and easily purify these cellular retinoid binding proteins from their cells of origin have been limited by a number of factors. First, the yields of purified protein have been poor: 15/xg of CRBP per gram wet weight of rat liver and 170/zg of CRBP II per gram wet weight of rat small intestine. 8,9 Second, copurification of comparably sized, homologous cellular retinoid-binding proteins may occur when several are expressed in a given tissue. Last, the purified proteins contain bound ligand. To analyze apoprotein-ligand interactions, UV irradiation or organic extraction is required to remove endogenous retinoids, methods which may perhaps lead to structural modifications that could complicate interpretation of subsequent functional studies. By contrast, purification of these mammalian proteins after their expression in Escherichia coli offers several advantages: (1) large quantities of protein are easily obtained; (2) only one retinoid-binding protein is expressed because E. coli does not possess any endogenous retinoid-binding proteins; (3) because E. coli does not require retinoids for growth and endogenous retinoids are not present, the retinoid-binding proteins purified from this prokaryote can be obtained as apoproteins; (4) site-directed mutagenesis of cloned cDNAs encoding retinoid-binding proteins and their subsequent expression in E. coli represent a powerful method for analyzing protein structure-activity relationships; and (5) amino acid derivatives can be introduced into the proteins by growing suitable E. coli auxotrophs containing a prokaryotic expression vector in the presence of the analog. Analog-substituted retinoid-binding proteins can, in turn, be used for nui M. M. Bashor, D. O. Tort, and F. Chytil, Proc. Natl. Acad. Sci. U.S.A. 70, 3483 (1973). 2 D. E. Ong and F. Chytil, J. Biol. Chem. 253, 828 (1978). 3 j. Sundelin, H. Anundi, L. Tragardh, U. Erikson, P. Lind, H. Ronne, P. A, Peterson, and L. Rusk, J. Biol. Chem. 260, 6488 (1985). 4 D. E. Ong, J. Biol. Chem. 259, 1476 (1984). 5 E. Li, L. A. Detainer, D. A. Sweetser, D. E. Ong, and J. I. Gordon, Proc. Natl. Acad. Sci. U.S.A. 83, 5779 (1986). 6 j. Sundelin, S. R. Das, U. Eriksson, L. Rask, and P. A. Peterson, J. Biol. Chem. 260, 6495 (1985). 7 D. A. Sweetser, R. O. Heuckeroth, and J. I. Gordon, Annu. Reo. Nutr. 7, 337 (1987). 8 D. E. Ong, A. J. Crow, and F. Chytii, J. Biol. Chem. 257, 13385 (1982). 9 W. H. Schaefer, B. Kakkad, J. A. Crow, I. A. Blair, and D. E. Ong, J. Biol. Chem. 264, 4212 (1989).
508
ENZYMOLOGY AND METABOLISM
[58]
clear magnetic resonance (NMR) studies of ligand-protein interactions (see below). Expression of Cellular Retinol-Binding Proteins in Escherichia coli General Principles For a general discussion of useful strategies for expressing, identifying, and characterizing recombinant gene products in E. coli, the reader is referred to the review by Shatzman and Rosenberg in this series.l° Two desirable components of prokaryotic expression vectors are (I) inducible promoters which can direct efficient transcription of foreign cDNAs and (2) translational control elements that allow efficient initiation of translation of the foreign mRNA transcript to occur. We have used pMON vectors 11 designed and provided by Monsanto z but a large number of commercially available expression vectors are also suitable. 12The pMON series of vectors are derived from pBR32713 and incorporate the E. coli recA promoter which can be induced by nalidixic acid and a ribosome binding site from the T7 bacteriophage gene 10 leader (G10L) sequence. II The pMON vectors that we have used contain a unique NdeI restriction site downstream from the recA promoter and the translational control element. By genetically engineering an NdeI site at its initiator ATG codon (if necessary), the cDNA of interest can be placed under the control of the recA promoter. Moreover, the distance between the vectorderived G10L ribosome-binding site and its translation start site is thereby maintained. One of the advantages of such a vector is that it directs the synthesis of a full-length "recombinant" polypeptide rather than one which represents a fusion between bacterial and eukaryotic protein sequences. The bacterial strain used for expression of the recombinant protein must be selected with several caveats in mind. First, the induction system used must be compatible with the bacterial phenotype. For example, a recA- strain cannot be used if the E. coli recA promoter is utilized. Second, E. coli proteases such as the product of the lon gene (protease La) may cause proteolysis of some foreign proteins. Expression of these proteins may be improved by using strains that are protease deficient (e.g., Lon- and Htpr-). Third, variables that can affect the efficiency of ~0A. R. Shatzman and M. Rosenberg, this series, Vol. 152, p. 661. H p. O. Olins, C. S. Devine, S. H. Rangwala, and K. S. Kavka, Gene 73, 227 (1988). lz Examples of currently available vectors that are suitable for production of "nonfusion" proteins in E. coli include pBTac2 and pBTrp2 (Boehringer Mannheim Biochemicals); pNHI8A (Stratagene); and pKK233-2 and pPL Lambda (Pharmacia LKB Biotechnology). 13 X. Sober6n, L. Covarrubias, and F. Bolivar, Gene 9, 287 (1980).
[58]
RAT CRBP AND CRBP II EXPRESSEDIN E. coli
509
production of recombinant proteins include incubation temperature, growth medium, cell density, timing of induction, and the length of fermentation after induction of foreign protein synthesis. Finally, it should also be noted that E. coli cannot support many critical posttranslational modifications of mammalian proteins (e.g., glycosylation). Growth o f Escherichia coli Transformed with p M O N - C R B P or pMON-CRBP H
We have not found it necessary to utilize protease-deficient hosts for the expression of CRBP and CRBP II. For most purposes, we use E. coli strain JM101 transformed with pMON-based vectors (Monsanto). A fresh overnight culture~ of E. coli JM101 containing the recombinant pMON vector is diluted 1 : 50 in fresh Luria broth plus ampicillin (100 /~g/ml) (pMON contains an A m p r locus). After achieving a cell density equivalent to an OD600 of approximately 0.2 nm, nalidixic acid is added (1:200 dilution of a 10 mg/ml stock prepared in 0.1 N NaOH) to activate the recA promoter of the plasmid. Fermentation is continued with agitation for an additional 3 hr at 30° (at which time the OD60o is -1.0). Cells are then harvested by centrifugation at 5,000 g or by filtration through a Millipore (Bedford, MA) Pellicon cassette (the Pellicon cassette is useful for harvesting cells from large-scale cultures, i.e., 20-100 liters). The cell paste is stored at - 7 0 °. Purification o f Escherichia coli Expressed Rat CRBP and CRBP H
Although foreign proteins that are expressed in E. coli may be soluble, many are present as insoluble aggregates known as inclusion bodies (reviewed by Marstonl4). Recovery of active proteins from these aggregates requires isolation of the inclusion bodies following cell lysis, subsequent denaturation to solubilize the protein, and finally a refolding reaction. Because soluble proteins can be purified directly, they are more likely to retain their native conformation. At the conclusion of the fermentation described in the preceding section, rat CRBP or CRBP II represents 1015% of the total soluble proteins present in E. coli homogenates. The purification of rat apo CRBP and apo CRBP II from E. coli is remarkably straightforward and relatively rapid, requiring two or t h r e e steps. 15,16Our protocol is summarized in Table I. An aliquot of the E. coli i4 A. Marston, Biochem. J. 240, 1 (1986). 15 E. Li, B. Locke, N. C. Yang, D. E. Ong, and J. I. Gordon, J. Biol. Chem. 262, 13773 (1987). t6 M. S. Levin, B. Locke, N. C. Yang, E. Li, and J. I. Gordon, J. Biol. Chem. 263, 17715 (1988).
510
ENZYMOLOGY AND METABOLISM
[58]
TABLE I PURIFICATIONOF RAT APo-CRBP AND APo-CRBP II FROMEscherichia coli LYSATES 1. 2. 3. 4.
5. 6. 7. 8. 9.
Thaw cell pellet and resuspend in 2-3 volumes cold lysis buffer (see text). Disrupt bacteria with a French press (-2000 psi). Clear lysate by centrifugation at 5000 g for 10 min (4°). Ammonium sulfate fractionation: Drip in an equal volume of cold filtered 100% saturated ammonium sulfate (Whatman) containing 15 mM dithiothreitol (pH adjusted to 7.1 with KOH) over 4 hr (keep on ice). Centrifuge as above to obtain a 50% ammonium sulfate supernatant. For CRBP II only, bring the 50% supernatant to 70% saturation by the addition of solid ammonium sulfate. Centrifuge as above to obtain the 50-70% pellet. Suspend this pellet in lysis buffer. Dialyze the ammonium sulfate fractions (-0.1-0.2 liters) overnight at 4° against -20 liters potassium phosphate (20 mM, pH 7.1), EDTA (1 mM), 2-mercaptoethanol (10 mM), sodium azide (0.05%), phenylmethylsulfonyl fluoride (0.02%, pH 7.1). Concentrate the postdialysis preparations, if necessary, to afinal protein concentration of - 5 mg/ml with an Amicon YMI0 membrane. Perform gel-filtration column chromatography with Sephadex G-50. Screen fractions by absorbance at 280 nm and by SDS-PAGE. Pool fractions containing CRBP or CRBP II. If the acyl cartier protein of E. coli is present (detected by SDS-PAGE and aminoterminal amino acid sequencing) or if separation of Met + from Met- CRBP is desired, then proceed to FPLC, Step 9. Perform FPLC with a Mono Q (Pharmacia) column equilibrated with imidazole (20 mM, pH 6.8) and eluted with a NaC1 gradient (see Fig. IB for details).
cell paste is first thawed to r o o m t e m p e r a t u r e and resuspended in 2 - 3 volumes of lysis buffer [Tris (final concentration 50 m M , p H 7.9), sucrose (10%), and p h e n y l m e t h a n e s u l f o n y l fluoride (0.5 mM)]. Bacteria are disrupted b y p a s s a g e through a F r e n c h Press ( - 2 0 0 0 psi). Cellular debris is r e m o v e d b y centrifugation at 5000 g for 10 min at 4 °. A m m o n i u m sulfate fractionation of the supernatant fraction is a v e r y useful first step in the purification: m o s t bacterial proteins are insoluble in solutions of NH4SO4 at 50% saturation, in contrast to the cellular retinol-binding proteins which remain soluble. Thus, the supernatant is adjusted slowly to 50% saturation with NH4SO4 (with constant stirring at 0-4°). After gentle stirring for an additional hour, the suspension is subjected to centrifugation at 41,000 g for 30 min at 4 °. An additional 70% NH4SO4 " c u t " is also beneficial w h e n purifying C R B P II. The o v e r 50% NH4SO4 supernatant containing C R B P or the 50-70% NH4SO4 precipitate containing C R B P II is then dialyzed overnight at 4 ° against buffer A [potassium p h o s p h a t e (20 m M , p H 7.1), E D T A (1 m M ) , glycerol (15%), sodium azide (0.05%), phenylmethanesulfonyl fluoride (0.5 m M ) , and 2 - m e r c a p t o e t h a n o l (10 mM)]. The dialyzed protein solution is then fractionated through a S e p h a d e x G-50-80 column equilibrated with the s a m e buffer. C o l u m n fractions are a s s a y e d for protein b y mea-
[58]
RAT C R B P AND C R B P II EXPRESSED IN E. coil
511
suring the absorbance at 280 nm. Selected fractions are then surveyed by electrophoresis through a 15% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS-PAGE). Typically, CRBP and CRBP II elute from the gel-filtration column at a relative retention volume (Ve/Vo) of 1.5 (see Fig. 1A). When purifying CRBP (but not CRBP II) an additional purification step is desirable to avoid contamination with the acyl carrier protein of E. coli. The acyl carrier protein is a soluble 8.8-kDa protein which migrates on SDS-PAGE as an approximately 20-kDa protein and elutes like a 12-kDa protein from Sephadex G-50 (reviewed by Rock and Cronan17). Those fractions which appear to be enriched for CRBP by SDS-PAGE are pooled and further purified by fast protein liquid chromatography (FPLC). The pooled fractions are applied to a Pharmacia (Piscataway, NJ) Mono Q column equilibrated with imidazole (20 mM, pH 6.8) and sodium azide (0.05%). CRBP is eluted with a continuous NaCI gradient (15.5-25 mM) in the same buffer. Two CRBP peaks elute at approximately 19 and 20 m M NaC1 (see Fig. 1B). These peaks correspond to CRBP without and with its initiator methionine residue, respectively. The proportion of Met + CRBP and Met- CRBP varies from protein preparation to preparation. The initiator methionine of foreign proteins expressed in E. coli may be removed coincidently, or shortly after synthesis, by a nonspecific aminopeptidase. ~8,19 The efficiency of methionine removal from recombinant proteins is variable and affected by the physicochemical properties of the penultimate amino acid. 18,~9The spectrofluorimetric and ligand binding properties of Met + and Met- CRBP appear to be identical. 16 For some applications, such as protein crystallization and NMR studies, it may be desirable to separate the two species (see below). Protein integrity and purity can be assessed by SDS-PAGE, isoelectric focusing, and automated sequential Edman degradation. Unlike the forms recovered from mammalian cells or cell-free translation systems (wheat germ or reticulocyte lysates), E. coli-derived rat CRBP and CRBP II do not have an acetyl group linked to their amino-terminal amino acid. 15,16Therefore, the intact E. coli-derived proteins can be sequenced directly. Purified E. coli-derived rat apo-CRBP and apo-CRBP II are stored in plastic tubes at 4° in phosphate buffer (20 mM, pH 7.4) supplemented with 2-mercaptoethanol (1 mM), EDTA (1 mM), and NaN3 (0.05%). We have not found aggregation to be a problem even at protein concentrations as high as 15 mg/ml ( - 1 mM). 17 C. O. Rock and J. E. Cronan, Jr., this series, Vol. 71, p. 341. 18 j. L. Brown, Biochim. Biophys. Acta 221, 480 (1970). 19j. L. Brown and J. F. Krall, Biochem. Biophys. Res. Commun. 42, 390 (1971).
512
ENZYMOLOGY AND METABOLISM
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