[49] Prokaryotic expression of catalytic subunit of adenosine cyclic monophosphate-dependent protein kinase

[49]

EXPRESSION OF c A P K CATALYTIC SUBUNIT

581

[49] P r o k a r y o t i c E x p r e s s i o n o f C a t a l y t i c S u b u n i t o f Adenosine Cyclic Monophosphate-Dependent Protein Kinase By WES M. YONEMOTO, MARIA L. MCGLONE, LEE W. SLICE,

and SUSAN S. TAYLOR cAMP-dependent protein kinase (cAPK) is a tetrameric holoenzyme composed of two catalytic and two regulatory subunits. Activation of cAPK occurs following the binding of cAMP to the regulatory subunit dimer and subsequent release of the monomeric catalytic subunits. Due to its small size, abundance, and relative ease of purification, the catalytic subunit of cAPK is one of the better characterized protein kinases in terms of the substrate requirements of the enzyme, the identification of residues involved in catalysis, and its mechanism of regulation.1 Protein kinases function as regulatory enzymes involved in signal transduction and cellular metabolism. 2The activity of these proteins, in general, is tightly regulated and directed toward specific cellular targets in vivo. Since protein kinases are usually expressed at low levels in cells, overexpression of eukaryotic protein kinases has been used to aid in the purification and characterization of their qualitative properties in oivo and in oitro. In addition, the production of milligram quantities of protein is necessary for the identification of functional amino acid residues by chemical modification as well as for structural analysis by physical methods such as nuclear magnetic resonance (NMR) and crystallography. 3 Three major eukaryotic expression systems have been utilized for the production of protein kinases: (1) mammalian cells, (2) baculovirus, and (3) yeast. Despite the importance and advantages of in vivo analysis of biological function and accurate posttranslational processing available with the use of mammalian cells, the level of protein expression is low relative to the other systems discussed here. 4 In addition, depending on the cell line used for expression, endogenous protein kinases may contaminate purified preparations of mutant recombinant enzyme. Baculovirus expression has been used for the overexpression of a variety of proteins with various degrees of success. 5-7 Higher levels of protein expression can be obtained compared to mammalian cells with reasonably similar posttransI S. S. Taylor, J. A. Buechler, and W. Y o n e m o t o , Annu. Rev. Biochem. 59, 971 (1990). 2 E. G. K r e b s , Enzymes 17, 3 (1986). 3 S. S. Taylor, J. A. Buechler, and D. R. Knighton, in " P e p t i d e s and Protein Phosphorylation" (B.E. K e m p , ed.), p. 1. Uniscience C R C Press, Boca Ration, Florida, 1990. 4 S. R. Olsen a n d M. D. Uhler, J. Biol. Chem. 264, 20940 (1989). 5 V. A. L u c k o w and M. D. S u m m e r s , Bio/Technology 6, 47 (1988).

METHODS IN ENZYMOLOGY. VOL. 200

Copyright © 1991by AcademicPress, Inc. All fights of reproduction in any form reserved.

582

ANALYSIS OF PROTEIN KINASES USING e D N A CLONES

[49]

lational modification. There is some technical difficulty in identifying recombinant viral plaques necessary for expression and this system cannot be used to analyze biological function in vivo. Overall, however, baculovirus represents an attractive system for protein production with the drawbacks of the extensive labor and expense inherently involved in standard tissue culture techniques. The expression of protein kinases in yeast represents one alternative to the use of tissue culture cells. Genetic manipulation of yeast cells can allow for expression of certain mammalian enzymes in the absence of expression of the analogous yeast enzyme. Interestingly, a number of studies have been described in which the expression of mammalian gene is able to complement a loss of the analogous yeast gene.S'9 Zoller and co-workers discuss in detail the methodology and optimization for the expression of protein kinases in yeast.l° In contrast to the eukaryotic systems described above, prokaryotic expression of foreign genes is a flexible and inexpensive system, using a variety of strong constitutive and inducible promoters. Milligram quantities of proteins can easily be produced for biochemical and physical studies with very little expense. In addition, expression in bacterial cells provides a method to produce recombinant eukaryotic proteins in the absence of endogenous wild-type enzymes. Finally, the complete lack of specific eukaryotic posttranslational modifications allows one to address the role of these modifications in the protein function. Expression in Escherichia coli is successfully used for high-level production of numerous eukaryotic proteins.ll,~2 Unfortunately there has been limited success for the expression of protein kinases in bacteria. Although the expression of many protein kinases leads to the production of milligram quantities of protein, it is mainly in an insoluble, inactive form, such as seen for pp60 src.13,14The expression of truncated protein kinases (such as p62 v-abl) can produce a

6 R. Herrera, D. Lebwohl, A. Garcia-de-Herreros, R. G. Kallen, and O. M. Rosen, J. Biol. Chem. 263, 5560 (1988). 7 H. Piwnica-Worms, N. G. Williams, S. H. Cheng, and T. M. Roberts, J. Virol. 64, 61 (1990). s T. Kataoka, S. Powers, S. Cameron, O. Fasano, M. Goldfarb, J. Broach, and M. Wigler, Cell (Cambridge, Mass.) 40, 19 (1985). 9 M. G. Lee and P. Nurse, Nature (London) 327, 31 (1987). 10 M. J. Zoller, K. E. Johnson, W. Yonemoto, and L. Levine, this volume [51]. 11 F. A. O. Marston, Biochem. J. 240, 1 (1986). ~2F. A. O. Marston, " D N A Cloning: A Practical Approach" (D. M. Glover, ed.), Vol. 3, p. 59. IRL Press, Oxford, 1987. 13 T. M. Gilmer and R. L. Erikson, Nature (London) 294, 771 (1981). 14j. D. McGrath and A. D. Levinson, Nature (London) 295, 423 (1982).

[49]

EXPRESSIONOF cAPK CATALYTICSUBUNIT

583

protein that can be solubilized with intact enzymatic activity. 15 Other eukaryotic protein kinases have been expressed in prokaryotic cells. This chapter will deal with the expression and purification of soluble and active cAPK catalytic (C) subunit in E. coli. Materials

Reagents are purchased as follows: Bacto-tryptone and Bacto-yeast extract bacterial media (Difco, Detroit, MI); ampicillin (sodium salt), Boeringer Mannheim (Mannheim, Germany); kanamycin monosulfate (Sigma, St. Louis, MO); P-11 phosphocellulose (Whatman, Clifton, N J); Q-Sepharose, CM-Sepharose, and Sephacryl-200 HR resins (Pharmacia/LKB, Piscataway, N J); isopropyl-fl-o-thiogalactopyranoside (IPTG), Sequenase, deoxy- and dideoxynucleotides (U.S. Biochemicals, Cleveland, OH). All chemicals not specifically cited are of reagent or molecular biology grade. All other DNA-modifying and restriction enzymes are obtained from Bethesda Research Laboratories (Gaithersburg, MD) or New England Bio Labs, Inc. (Boston, MA). The following bacterial strains are used: E. coli RZ1032 (ATCC); E. coli DH5a (Bethesda Research Laboratories); E. coli BL21(DE-3) (W. Studier, Brookhaven National Laboratory, Upton, NY). Prokaryotic Expression of C~ Subunit of cAMP-Dependent Protein Kinase Slice and Taylor 16have described the expression of a cDNA clone for the murine C~ subunit of cAPK 17in bacterial cells. A number of different expression vectors and bacterial strains were tested for their ability to express the C~ protein. These vectors included expression of the C~ cDNA from the lacUV5 promoter of pUC18 as well as coexpression with the cDNA of the wild-type bovine RI subunit. TM Although expression was obtained from the different C~ subunit-producing E. coli cells examined, in general, the recombinant protein produced was inactive and found in insoluble inclusion bodies. 19 The expression of the C~ subunit from the bacteriophage T7 gene l0 15 j. y . j. Wang, C. Queen, and D. Baltimore, J. Biol. Chem. 257, 13181 (1982). 16 L. W. Slice and S. S. Taylor, J. Biol. Chem. 264, 20940 0989). 17 M. D. Uhler, D. F. Carmichael, D. C. Lee, J. C. Chrivia, E. G. Krebs, and G. S. McKnight, Proc. Natl. Acad. Sci. U.S.A. 83, 1300 (1986). Is L. D. Saraswat, M. Filutowics, and S. S. Taylor, J. Biol. Chem. 261, 11091 (1986). 19 N. J. Darby and T. E. Creighton, Nature (London) 334, 715 (1990).

584

ANALYSIS OF PROTEIN KINASES USING c D N A CLONES

[49]

(~bl0) promotor 2° in E. coli BL21(DE-3) made significant amounts of soluble and active Ca subunit. Escherichia coli BL21(DE-3) contains the bacteriophage T7 gene I (T7 RNA polymerase) under inducible control of the lacUV5 promotor. 2~Following addition of IPTG, the expression of T7 RNA polymerase directs the expression of the Co gene. When Ca expression was carried out at 37°, approximately l0 to 20% of the protein expressed was found in the soluble fraction. This finding was significant since it provided a method for the production of large amounts of recombinant Ca subunit with relative ease at little expense. In addition, this system provides a method with which to produce mutants of the C subunit for analysis of their biochemical and physical properties. Interestingly, the use of E. coli BL21(DE-3) was important for the production of soluble enzyme, for when the same T7 gene 10 promotor/ Ca cDNA expression plasmid vector (pLWS-3) ~6was expressed in E. coli K38, only insoluble C subunit was produced. Escherichia coli K38 contains the bacteriophage T7 RNA polymerase under control of a heatinducible h PL promotor and requires elevated temperature for gene expression. 2° It seems likely that this elevation in temperature during expression is at least partly responsible for the insolubility of the recombinant Ca protein (see below), although other variations between the two T7 expression E. coli strains may account for the difference in the ability to produce soluble recombinant protein. In order to make oligonucleotide site-directed mutants, the Ca cDNA is subcloned into the phagemid vector, pUC 11922and single-stranded DNA template is prepared as previously described in E. coli RZ1032. 23Mutagenesis protocols are performed essentially as described by Zoller and Smith 24 and the mutants are verified by dideoxy-DNA sequencing with Sequenase (U.S. Biochemicals). Mutant Ca genes are resubcloned back into the multiple cloning site of the plasmid vector pT7-7, downstream from the bacteriophage T7 gene 10 promoter. 2° In order to obtain expression of the Ca cDNA, the pT7/C, plasmid vectors are transformed into the E. coli BL21(DE-3) as previously described. 16 Colonies are screened for expression of Ca protein after growth in YT medium (8 g Bacto-tryptone, 5 g Bacto-yeast extract, 5 g NaC1/liter) with ampicillin (100/.~g/ml) followed by induction with 0.4 mM IPTG for 3 hr at 37°. Total crude cell extracts are prepared by lysing the cells in boiling SDS sample buffer and analyzed ~0 S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. U.S.A. 82, 1074 (1985). 21 F. W. Studier and B. A. Moffatt, J. Mol. Biol. 189, 113 (1986). 22 j. Viera and J. Messing, this series, Vol. 153, p. 3. 23 T. A. Kunkel, J. D. Roberts, and R. A. Zakour, this series, Vol. 154, p. 367. 24 M. J. Zoller and M. Smith, this series, Vol. 154, p. 329.

[49]

EXPRESSION OF cAPK CATALYTICSUBUNIT

MW C-SUB.

585

I-

m

m

A

B

C

D

E

FIG. 1. Expression of wild-type and mutant Ca proteins in total bacterial cell extracts. Total extracts ofE. coli BL21(DE-3) cells expressing C, proteins were prepared as described in the text and analyzed by SDS-PAGE: (A) wild-type C~ subunit, (B) C~(Cys343Ser)subunit, (C) C~(Lys72Arg) subunit, (D) C~(Lys285stop) subunit, (E) BL21(DE-3) cells.

by SDS-PAGE. 25 Figure 1 shows the Coomassie Blue-stained SDS gel of a number of different recombinant Ca proteins. Lane A (Fig. 1) shows the relative expression of wild-type recombinant Ca subunit in the total bacterial lysate following induction with IPTG, while lanes B, C, and D (Fig. 1) show the relative level of expression of various mutant Ca proteins expressed in BL21(DE-3) cells. Lane E (Fig. 1) is a sample of total bacterial lysate from the parental BL21(DE-3) cells. It is important to screen a number of different BL21(DE-3) transformants for expression of recombinant Ca subunit since we have noticed variations in the level of Ca expression in different colonies. Those bacterial colonies which express high levels of C a subunit are grown to midlog phase (OD 0.5 to 0.6 at A600). The cells are harvested by gentle centrifugation and resuspended in an equal volume of 2YT 25 U. K. Laemmli, Nature (London) 227, 680 (1970).

586

ANALYSIS OF PROTEIN KINASES USING c D N A CLONES

[49]

medium (16 g Bacto-tryptone, 10 g Bacto-yeast extract, 5 g NaCl/liter) containing 50% (v/v) glycerol (no antibiotics). The cell suspension is then divided into 200-/zl aliquots in 1.5-ml Eppendorf tubes and frozen quickly over dry ice. The frozen bacterial stocks can be stored at - 7 0 ° indefinitely. We have found it is important not to grow the stock cell cultures to late log or stationary phase since the growth of cells producing the highest levels of C~ subunit tends to be unfavorable under those conditions.

Effect of Temperature on the Production o f Catalytic Subunit Previous studies have indicated that temperature can affect the solubility of proteins produced in E. coli. 26 This finding is of interest since many eukaryotic protein kinases produced in bacterial systems are made in an insoluble, inactive form. Generally only the fraction of soluble recombinant protein kinase, or enzyme resolubilized from the bacterial insoluble fraction, possesses functional kinase activity. Therefore, the effect of temperature on the solubility of the recombinant C~ subunit was examined. BL21(DE-3) cells containing the wild-type C~ gene (pLWS-3) were grown at 37° in YT medium with ampicillin (100 tzg/ml). When the cell culture reached an OD of 0.8 at A600, IPTG was added to a final concentration of 0.4 mM. The culture was divided into three samples which were individually shaken and incubated for 4 hr at the temperature indicated. After the indicated period, the cells were harvested and lysed using a French pressure cell. The cell lysates were separated into soluble and particulate fractions by differential centrifugation at 17,000 g for 30 min. The soluble and particulate fractions were collected and samples were analyzed by SDS-PAGE and then stained with Coomassie Blue. Figure 2 shows the analysis of the effect of temperature on the solubility of the recombinant C subunit. At 37°, a majority of the C~ subunit was found in the insoluble, particulate fraction of the cell lysate. At 31°, roughly 50% o f t h e C~ subunit produced was observed in the soluble fraction. At room temperature (24°) essentially all of the C~ subunit produced was found in the soluble fraction of the cell lysate. The overall level of production of the C~ subunit was lower at 24 versus 37°, which reflects a slower rate of protein synthesis. Therefore, as the incubation temperature during induction was lowered, a greater proportion of the C~ subunit produced was found in the soluble fraction of the cell lysate.

C. H. Schein and H. M. Noteborn,

Bio/Technology 6, 291 (1988).

[49]

EXPRESSION OF cAPK CATALYTICSUBUNIT

37 ° M W CAT

P

587

31 ° S

P

24 ° C S

P

S

200 97 68 43

29 FIG. 2. Effect of temperature on solubility of the Ca subunit. Wild-type Ca subunitexpressing E. coli BL21(DE-3) cells were grown and induced under different temperature conditions as specifiedin the text. The bacterial cells were harvested, lysed, and fractionated into particulate (P) and soluble (S) by differential centrifugation. The samples induced at 37, 31, and 24° were analyzed by SDS-PAGE. Molecular weight (x 10-3).

Effect o f Induction Time on Production o f Catalytic Subunit Studier and Moffat have described the effect of induction time upon R N A and protein production under the control of bacteriophage T7 promoters in E. coli BL21(DE-3) cells. 21 At 37 °, we have found maximal production of C~ subunit within 3 to 4 hr after the addition of IPTG and induction o f bacteriophage T7 R N A polymerase. Since production of soluble C~ subunit was favorable at lower temperatures, the time course of protein synthesis and accumulation was reexamined at 24 ° . The pT7/C~-BL21(DE-3) cells were grown in Y T with ampicillin until an OD at A600 o f 0.8 was reached, at which time the temperature was shifted to 24 ° and the cultures were induced by the addition of IPTG (0.4 mM). Samples were taken at the times indicated, harvested by centrifugation in a microfuge, and resuspended in SDS sample buffer. The cells were lysed by boiling and analyzed by S D S - P A G E and Coomassie Blue staining. Figure 3 shows the results of the induction time course. Detectable levels of the C~ subunit continued to accumulate until 6 to 8 hr after induction. After 8 hr, no change in the level o f C~ subunit was observed up to 24 hr

588

ANALYSIS OF PROTEIN KINASES USING c D N A CLONES

[49]

T I M E / HOURS M

0

1

2

4

8

12

CAT. P"

FIG. 3. Time course of expression of the C~ subunit. Wild-type Ca subunit-expressing E. coli BL21(DE-3) cells were grown and then induced at 25°. Samples were removed at the times indicated and samples were prepared and analyzed by SDS-PAGE as described in the text.

(data not shown). Therefore the use of lower temperature during induction requires a concomitant lengthening of the time required for protein synthesis and accumulation in order to achieve production of maximal levels of the soluble recombinant C~ subunit. Purification of the C~ Subunit When cAPK is purified from mammalian cells or tissues, it is generally first isolated as holoenzyme complex on an anion-exchange resin, such as DEAE-Sepharose. 27 Following dissociation of the holoenzyme with the addition of cAMP, the C subunit is typically purified to homogeneity by CM-Sepharose chromatography. 2s Hydroxyapatite 29 and Blue dextran M. J. Zoller, A. R. Kerlavage, and S. S.Taylor, J. Biol. Chem. 254, 2408 (1979). 2s N. C. Nelson and S. S. Taylor, J. Biol. Chem. 256, 3743 (1981). 29 E. M. Reimann and R. A. Beham, this series, Vol. 99, p. 51.

[49]

EXPRESSIONOF cAPK CATALYTICSUBUNIT

589

chromatography 3° have also been used to purify the C subunit. Initial attempts at purification of the recombinant Ca subunit from the crude cell supernatant by standard anion, cation, hydrophobic, and hydroxyapatite chromatography all failed, possibly due to the aggregation of the C subunit with bacterial proteins and/or nucleic acids. Purification of the recombinant Ca subunit was finally achieved by the use of two novel protocols involving (1) the cation-exchange resin, phosphocellulose, and (2) the addition of a recombinant double mutant of the RI subunit which forms holoenzyme in the crude bacterial lysate.

Phosphocellulose Chromatography~Gel Filtration In order to purify the Ca subunit, 12 liters of E. coli BL21(DE-3) cells transformed with a pT7/C~ plasmid are grown overnight at 37° in YT medium containing 100/xg/ml ampicillin. When the OD at Ar00 reaches between 0.8 and 1.0, IPTG is added to a final concentration of 0.4 mM and the cells are grown for an additional 8 to 10 hr at 24 °. The bacterial cells are harvested, resuspended in 260 ml buffer A [30 mM MES, pH 6.5, 50 mM KCI, 1 mM EDTA, 5 mM 2-mercaptoethanol(2-ME)] and lysed in a cold (4°) French pressure cell. Protease inhibitors may be added to the lysis buffer, but the recombinant Ca subunit does not appear to be very sensitive to proteolysis provided the lysate is kept cold (4°) at all subsequent purification steps. The cell lysate is clarified by centrifugation at 17,000 g for 30 min and the pellet is discarded. The first purification method utilizes P-11 phosphocellulose and gelfiltration chromatography. This procedure can be used for wild-type recombinant Ca subunit, as well as for a number of mutant Ca subunits. P-11 phosphocellulose resin is prepared according to the manufacturer's instructions. Briefly, 15 g of resin is swollen in a 50 x volume of 0.5 M NaOH for exactly 5 min. The resin is washed extensively with H20 in a sintered glass funnel until the eluate was at pH 11, and then is transferred into a 50 x volume of 0.5 M HCI for 5 min. The resin is again washed with H20 until the eluate is at pH 3.0. The pretreated resin is washed with excess 300 mM MES, pH 6.5 until the washes equilibrate at pH 6.5. The resin then is poured into a column [3 cm (diameter) x 23 cm (length)] and preequilibrated with buffer B (30 mM MES, pH 6.5, 1 mM EDTA). This is a very important step since the recombinant Ca subunit binds over a very narrow pH range to phosphocellulose. The supernatant from the cell lysate is diluted with cold H20 to a conductivity of 1.1 ml-I and applied to the prepared 100 ml of P-11 column. After extensive washing with buffer 3o j. j. Witt and R. Roskoski,

Biochemistry 14, 4503 (1975).

590

ANALYSIS OF PROTEIN KINASES USING c D N A CLONES

[49]

A

E 0.4

In 0.3

at O z ,<

0.2

m

0

m m <

0.1

8

16

24

32

40

48

56

64

FRACTION NUMBER FIG. 4. (A) Phosphocellulose chromatography of C~ subunit. The column (3 x 23 cm) was packed with 15 g of prepared P-11 phosphocellulose equilibrated in buffer B. Twelve liters of E. coil BL21(DE-3) cells expressing C~(Cys343Ser) subunit was processed as described in the text. The C~ subunit was eluted with a linear KPO4 gradient (50-400 mM) and fractions of 3 ml were collected. Selected fractions were also subjected to SDS-PAGE (inset). (B) Sephacryl-200 HR chromatography of C~ subunit. The fractions containing C~(Cys343Ser) subunit after phosphocellulose chromatography were pooled and concentrated by ammonium sulfate precipitation as described in text. The column (3 x 120 cm) was equilibrated with buffer C and eluted with a flow rate of 2 ml/min. Fractions of 3 ml were collected and the C~ protein was detected by SDS-PAGE (inset).

B, the proteins are eluted with a 300-ml linear gradient from 50 to 400 mM KPO4, pH 6.5. Figure 4A shows the elution profile and SDS-PAGE analysis after P-11 chromatography for the mutant Cys343Ser C,~ protein. This mutant protein behaves identically to the wild-type C, subunit during phosphoceUulose chromatography. The Cys343Ser C a subunit is identified by its migration on the SDS-PAGE and by spectrophotometric protein kinase activity assays with the substrate peptide, kemptide. 3~ The P-11 fractions containing the Co subunit are pooled and concentrated by precipitation with the addition of solid ammonium sulfate to a 31 R. Roskoski, this series, Vol. 99, p. 1.

[49]

EXPRESSIONOF cAPK CATALYTICSUBUNIT

591

B

E C

0.4

'd" In o4

0.3--

O Z .<

0.2_

m re.

O m

0.1

m,

< , -

-

,

8

_'

;J~r~l 16 24

32

40

48

56

64

FRACTION NUMBER FIG. 4. (continued)

final concentration of 80%. The precipitate is collected and redissolved in approximately 2.0 ml of buffer C (25 mM KPO4, pH 7.0, 200 mM KCI, 5 mM 2-mercaptoethanol) and applied to a Sephacryl-200 HR column equilibrated with buffer C. The C~ subunit is eluted with 300 ml of buffer C, pumped at a rate of 2 ml/min. Figure 4B shows the elution profile and SDS-PAGE analysis after gel-filtration chromatography. One major peak of protein elutes from the column. This protein is confirmed to be the C~ subunit by its migration on SDS-PAGE, Western blot analysis with antibodies directed against the C~ subunit, and in vitro protein kinase assays.

Holoenzyme Purification with RI Subunit Although phosphocellulose chromatography provides an effective purification for the wild-type and some of the mutant recombinant C~ proteins, other mutant C~ subunits do not bind to P-11 directly from the crude bacterial lysate. Most of these mutant C~ subunits contain point mutations of highly conserved residues within the catalytic core region shared by all known eukaryotic protein kinases. 32Therefore a second and novel proto32 S. K. Hanks, A. M. Quinn, and T. Hunter, Science 241, 42 (1988).

592

ANALYSIS OF PROTEIN KINASES USING c D N A CLONES

[49]

col was devised to aid in the purification of recombinant Ca proteins. This procedure employs the use of a mutant R subunit with an altered affinity for cAMP. This mutant R subunit contains a Lys residue at a single conserved Arg position within each tandem cAMP-binding domain. 33This double RI subunit mutant, RI(ArgE°gLys : Arga33Lys),results in an increase of two orders of magnitude in the concentration of cAMP required for activation of the holoenzyme complex. This change in the affinity for cAMP allows the mutant RI subunit to bind to the catalytic subunit and form homoenzyme in the crude bacterial lysate. Wild-type RI subunit fails to form holoenzyme in crude cell lysates, due to the presence of endogenous cAMP. In order to purify the recombinant Ca subunit by this alternative procedure, 12 liters of Ca-expressing cells are harvested by centrifugation and combined with the bacterial pellet from 6 liters of E. coli 222 expressing the RI(ArgE°gLys : Arga33Lys) subunit. The cells are lysed together in 300 ml buffer A (without EDTA) using a French pressure cell. Holoenzyme formation occurs immediately upon lysis as monitored by cAMP-dependent protein kinase activity. The cell lysate is centrifuged at 17,000 g for 40 min at 4 ° and the supernatant is collected. The conductivity is adjusted to I. 1 mX) with cold H20 and the diluted supernatant is applied to a column prepared with 100 ml of Q-Sepharose equilibrated in buffer D (10 mM MES, pH 6.5, 5 m M 2-mercaptoethanol). The column is washed extensively and eluted with a 500-ml linear gradient from 0 to 250 mM NaC1 in buffer D. The fractions containing holoenzyme as monitored by SDS-PAGE and protein kinase activity are pooled and diluted to a conductivity of 1.1 ml) with cold H20. It has previously been shown that the C subunit binds well to CMSepharose, while the R subunits and the holoenzyme are not retained. 28 Therefore, the diluted, pooled Q-Sepharose-purified holoenzyme fractions are first passed over a 100-ml CM-Sepharose column, equilibrated in buffer E (17 mM KPO4, pH 6.5, 5 mM 2-mercaptoethanol) in order to remove the bacterial proteins which bind to the resin. The flow-through fractions are collected and cAMP is added to a final concentration of 1 mM to dissociate the unbound holoenzyme complex. The dissociated holoenzyme is then reapplied to a fresh 100-ml column of CM-Sepharose equilibrated with buffer E, washed extensively with buffer E, and eluted with a 300-ml linear gradient from 0 to 400 mM NaCl in buffer E. Figure 5 shows the results of the CM-Sepharose purification of the Ca subunit. The peak fractions containing the Ca subunit are pooled and assayed for protein kinase activity. 13 I. T. Weber, T. A. Steitz, J. Bubis, and S. S. Taylor,

Biochemistry 26, 343 (1987).

[49]

EXPRESSION OF c A P K CATALYTIC SUBUNIT

593 ~J

68 66

r,J o O

o

w

64 62 60

"O'O ,.,~ J:::

58 56 54 52

=~:~

50 48 46 oo

:::

42 40

j::

O

O

38 36

>, ¢) "~

o= [ " ~

•

I

WASH FLOW LOAD

R-STD C-STD

--=

=o¢)

594

ANALYSIS OF PROTEIN KINASES USING c D N A CLONES

[49]

Approximately 20 to 40 mg of C a subunit is purified from 12 liters of bacterial cells by either the phosphocellulose or holoenzyme purification methods described above. The bacterial cells can be grown, induced, harvested, and frozen as a solid paste at - 7 0 ° for an indefinite period of time without any loss in yield of purified Ca subunit. A 12-liter preparation should take between 3 and 4 days from bacterial pellet to purified enzyme. A single operator can easily complete two purification runs in 1 week. Analysis of Properties of Recombinant C= Subunit Kinetic Parameters An initial characterization of the properties of the purified recombinant murine C= subunit compared to the C subunit protein purified from porcine heart was completed. 16Both the recombinant and mammalian C subunits were able to form holoenzyme with the R I subunit and the cAMP activation of the holoenzymes was indistinguishable. Both enzymes possess similar kinetic parameters with respect to the binding of the cosubstrates MgATP and the peptide, kemptide. The apparent K m values for MgATP and for kemptide were approximately 15 and 45/.~M, respectively, for both the recombinant and mammalian enzyme. In addition, both proteins were efficiently inhibited by the 20-residue inhibitor peptide 34 with an apparent Ki of approximately 12 nM. These results indicate that the recombinant C~ subunit produced in E. coli has roughly the same enzymatic properties as the protein isolated from mammalian tissues. One clear distinction between the recombinant and mammalian C subunit was the thermostability of the enzymatic activity to heat denaturation. There is approximately a 5° difference in the temperature required to inactivate 50% of the recombinant murine C~ subunit after a 2.5-min incubation compared to the mammalian porcine enzyme. It is presently unclear why this difference in thermostability between these two forms of the C subunit exists; however, it has been proposed that myristoylation of the N terminus may influence the stability of the recombinant protein. 16 Posttranslational Modifications The mammalian C subunit has been shown to contain two types of posttranslational modification: (1) phosphorylation 35 and (2) myristoylation.36 Two sites of phosphorylation were detected by amino acid sequenc34 D. A. Walsh, K. L. Angelos, S. M. Van Patten, D. B. Glass, and L. P. Garetto, "Peptides and Protein Phosphorylation" (B.E. Kemp, ed.). Uniscience CRC Press, Boca Raton, Florida, 1990. 33 K. A. Peters, J. G. Demaille, and E. H. Fisher, Biochemistry 16, 5691 (1977). 36 S. A. Carr, K. Biemann, S. Shoji, D. C. Parmalee, and K. Titani, Proc. Natl. Acad. Sci. U.S.A. 79, 6128 (1982).

[49]

EXPRESSION OF c A P K CATALYTIC SUBUNIT

595

ing of bovine C subunit, one at Thr-197, the other at Ser-338. 37In addition, the porcine C subunit has been shown to contain one major site of in vitro phosphorylation at Ser-10. 3a In vivo labeling of wild-type C~ subunit-expressing E. coli cells shows one major phosphoprotein which exactly comigrates with the C subunit. This protein was demonstrated to be the C~ subunit after purification by P-11 phosphocellulose chromatography, immunoblotting with polyclonal antibodies, and protein kinase activity assays. Phosphoamino acid analysis of the in vivo labeled recombinant C,~ subunit identifies the presence of both phosphoserine and phosphothreonine. Preliminary sequence analysis of the high-performance liquid chromatography (HPLC)-purified phosphotryptic peptides indicates that both Thr-197 and Ser-338, residues previously identified in the mammalian C subunit, are also phosphorylated in the recombinant enzyme. The amino terminus of the mammalian C subunit is blocked by the addition of a myristoyl group. Although the C subunit was the first protein discovered with this unique fattY acid modification, the biological function of this N-terminal myristoylation of the C subunit is not known. 36 Subsequently this posttranslational modification has been found on other cellular and viral proteins and in some cases has been shown to be necessary for association with membranes.39 Since myristoylation is a eukaryotic protein modification, it was not expected that the recombinant C~ subunit would be acylated. Amino acid sequencing of the recombinant C~ subunit revealed a free N-terminal Gly residue. 16 Therefore, the major physical difference between the recombinant and mammalian enzymes, detected so far, is the lack of an N-terminal myristoylation. Gordon and co-workers have cloned the gene for the yeast N-terminal myristoyltransferase and have shown that prokaryotic expression of the gene generates an active recombinant enzyme. Coexpression of the transferase together with the C~ subunit leads to acylation of the recombinant C~ subunit in vivo, thereby reconstituting this posttranslational modification system in E. coli. 4° Expression and purification of recombinant, myristoylated C~ subunit allows investigation of the physical properties of the modified C~ subunit. Specifically, the role of myristoylation in the thermostability of the recombinant C,~ subunit can directly be readdressed. 37 S. Shoji, K. Titani, J. G. Demaille, and E. H. Fisher, J. Biol. Chem. 254, 6211 (1979). 38 j. Toner-Webb and S. S. Taylor, J. Biol. Chem. (submitted for publication). 39 D. A. Towler, S. R. Eubanks, D. S. Towery, S. P. Adams, and L. Glaser, J. Biol. Chem. 262, 1030 (1987). ~0 R. J. Duronio, E. Jackson-Machelski, R. O. Heuckeroth, P. O. Olins, C. S. Devine, W. Yonemoto, L. W. Slice, S. S. Taylor, and J. I. Gordon, Proc. Natl. Acad. Sci. U.S.A. 87, 1506 (1990).

596

ANALYSIS O F PROTEIN KINASES USING c D N A

CLONES

[50]

Summary The prokaryotic expression of the C~ subunit of cAPK provides a system for the production of milligram quantities of wild-type and mutant protein with relative ease and little expense. The protocols described here have been optimized for the production of soluble and active protein kinase, with kinetic parameters similar to those found for the enzyme isolated from mammalian tissue. The C~ subunit expressed in E. coli is a phosphoprotein and appears to contain the sites of phosphorylation identified in the mammalian protein. The free N-terminal Gly indicates the lack of an N-terminal myristic acid; however, coexpression of the gene encoding the yeast N-myristoyltransferase allows for myristoylated C~ subunit to be produced in E. coli. Finally, the purified recombinant C~ subunit has been used in X-ray crystallographic studies which have yielded the crystals that will allow the first three-dimensional structure of a protein kinase to be solved. Therefore, although the employment of prokaryotic expression in the production of functional protein kinases has been limited, it is hoped that the methods presented here for the C subunit of cAPK will encourage the use of this simple and versatile expression system. Acknowledgments This work was supported in part by the AmericanCancer Society Grant BC-678E to S.S.T.W.M.Y. is a postdoctoralfellowof the CaliforniaDivisionof the AmericanCancer Society.

[50] E x p r e s s i o n a n d P u r i f i c a t i o n o f A c t i v e a b l P r o t e i n T y r o s i n e K i n a s e in E s c h e r i c h i a coli By SYDONIA

I. RAYTER

Protein-tyrosine kinase activity is the functional enzyme activity associated with many transforming retroviruses (such as those encoding src and abl) and growth factor receptors (such as epidermal growth factor, platelet-derived growth factor, insulin, insulin-related growth factor-1, and colony-stimulating factor- I). Approximately 30 proven or putative proteintyrosine kinases have been identified, either as the transforming protein of retroviruses, the activity associated with growth factor receptors, by METHODS IN ENZYMOLOGY, VOL. 200

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.