- Email: [email protected]
[1]The use of melittin-sepharose chromatography for gram-preparative purification of calmodulin
Ill
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
3
[1] T h e U s e o f M e l i t t i n - S e p h a r o s e C h r o m a t o g r a p h y for Gram-Preparative Purification of Calmodulin By RANDALL L. KINCAID
Introduction
The successful application of affinity chromatography relies on the specific retention of macromolecules on a matrix of immobilized ligand. Ideally, the properties of the matrix should meet several criteria. First, the interaction should be of high affinity (Kd - 10 -7 M ) to enable rigorous washing of the gel without desorption of bound material. Second, the association of bound proteins with ligand should be readily reversed by addition of an eluting agent which either competes for ligand (substrate competition) or which greatly reduces the affinity of the ligand-macromolecule complex. Last, the properties of the matrix should be well defined, permitting quantitative analysis, and stable to repeated usage and regeneration procedures. With these considerations in mind, we prepared an affinity matrix of immobilized melittin for purification of calmodulin (CAM). z Melittin, the major component of bee venom, is a 26 amino acid peptide which is amphipathic; it contains a long stretch of hydrophobic residues, a single aromatic residue (tryptophan, position 19) and several positively charged side chains clustered near the carboxyl terminus (Fig. 1). This peptide, which aggregates into tetramers at concentrations of 0.2 mg/ml and greater, 2 is soluble in both aqueous and lipid environments; as a consequence, melittin may have a general biological role as a surfactant. 3 Interestingly, several laboratories noted that mdittin inhibited cyclic nucleotide phosphodiesterase 4,5 and reported that inhibition was dependent on Ca 2+ and competitive with CaM. Later, physical studies documented direct stoichiometric interaction of melittin with CaM by noting changes in its fluorescence anisotropy 6,7 and by quantifying complexes formed during electrophoresis and gel filtration chromatography. 5 In I R. L. Kincaid and C. C. Coulson, Biochem. Biophys. Res. Commun. 133, 256 (1985). 2 T. C. Terwilliger and D. Eisenberg, J. Biol. Chem. 257, 6010 (1982). 3 E. Kn6ppel, D. Eisenberg, and W. Wickner, Biochemistry 18, 4177 (1979). 4 M. S. Barnette, R. Daly, and B. Weiss, Biochem. Pharmacol. 32, 2929 (1983). 5 M. Comte, Y. Maulet, and J. A. Cox, Biochem. J. 209, 269 (1983). 6 y . Maulet and J. A. Cox, Biochemistry 22, 5680 (1983). 7 D. A. Malencick and S. R. Anderson, Biochemistry 23, 2420 (1984).
METHODS IN ENZYMOLOGY, VOL. 139
4
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS
/ ~
NH 2
.
'.p
[1]
/J-~
A
'"
)
FIG. l. Schematicrepresentationof the aminoacid sequenceof melittin. The numbered circles indicate successiveaminoacids beginningwith the aminoterminus. The projections represent the carbon bonds in the side chains of each amino acid; positive charges are indicated for the lysineresidues at positions 7, 20, and 22 and for arginine at positions 21 and 23. those studies and from work in our own laboratory (R. Kincaid, unpublished data), the affinity of melittin for CaM was estimated to be in the nanomolar range. Because of this high binding constant, as well as its well-defined properties and availability, it seemed an appropriate ligand for the Ca2+-dependent affinity chromatography of CaM and perhaps other Ca2÷-binding proteins. Our studies have shown that melittinSepharose is suitable for large-scale purification of CaM and S-I00 protein from brain and that, in some tissues, purification of CaM to an essentially homogeneous state can be accomplished in a single step. The present chapter outlines analytical and preparative details for use of this affinity matrix. Preparation of Melittin-Sepharose (10 mg Melittin/ml Gel)
Materials 15 g CNBr-activated Sepharose 4B (Pharmacia) 4 liters HCI, 1 mM 0.2 liter coupling buffer (0.1 M sodium bicarbonate, pH 8.5, containing 0.5 M NaC1) 500 mg melittin, 10 mg/ml, dissolved in coupling buffer (Sigma or U.S. Biochemical Corp.) 75 ml 1 M ethanolamine- HCI, pH 8.0 0.2 liter low-pH urea regeneration buffer (0.1 M sodium acetate, pH 4.5, containing 6 M urea, I0 mM EDTA, and 0.5 M NaCI) 0.2 liter high-pH wash buffer (0.1 M sodium borate, pH 9.0, containing 0.5 M NaCI)
[1]
MELITTIN--SEPHAROSE AFFINITY CHROMATOGRAPHY
5
Procedure Step 1: Washing of CNBr-Sepharose 4B. The lyophilized CNBrSepharose 4B is swollen for 5 min in 100 ml 1 mM HC1 with gentle mixing. It is then transferred to a l-liter sintered glass funnel (coarse frit), resuspended in 400 ml 1 mM HCI, and filtered with suction to form a moist cake. The gel is suspended and filtered three more times, after which the gel cake (45-50 ml, bed volume) is transferred to a beaker and mixed with 25 ml i mM HC1. Step 2: Coupling of Melittin to CNBr-Sepharose 4B. To the washed gel, 50 ml of melittin (10 mg/ml) dissolved in coupling buffer is added and mixed immediately. To this, 7.5 ml each of 1 M sodium bicarbonate, pH 8.5, and 5 M NaCl are added to make final concentrations of 0.1 and 0.5 M, respectively. The slurry is transferred to a 250-ml plastic tissue culture flask (Falcon) and mixed on a tilting rocker (e.g., Thermolyne clinical test tube mixer). Step 3: Blocking of Excess Reactive Groups on Melittin-Sepharose. After 2 hr at room temperature, the suspension is suctioned on a 350-ml sintered glass funnel and the moist cake washed with 75 ml coupling buffer. The gel is transferred to a beaker and 25 ml coupling buffer is added. To this slurry 75 ml of 1 M ethanolamine- HCI, pH 8.0, is added and the suspension mixed for 2 hr as in step 2. Step 4: Washing and Storage of Melittin-Sepharose. After blocking, the gel is transfered to a 350-ml sintered glass funnel and suctioned. The gel is washed with 100 ml of low pH urea regeneration buffer, followed by 100 ml high pH wash buffer; this washing cycle is repeated. The gel is suspended in 50 ml of 20 mM 2-[bis-2[hydroxyethyl)amino]ethanesulfonic acid (BES) (Calbiochem), pH 7.2, containing 100 mM NaCl and 0.02% Thimerosal (Sigma) and stored at 4 °. Notes on Procedure. Step 1: Sepharose can also be activated using 150 mg CNBr/ml Sepharose 4B, at alkaline pH, as described, a Step 2: A substituent density of 5 mg melittin/ml gel is achieved by adding one-half the amount (i.e., 25 ml) of melittin solution. Step 3: The use of a low-pH urea-containing buffer is important to remove some colored, hydrophobic impurities. Optimization and Quantitative Properties of the Affinity Matrix An important step in preparation of an affinity matrix is to establish the optimum relationship between substituent concentration on the gel and binding capacity/recovery. Clearly, it would be best to achieve a high enough binding capacity to ensure a concentrated affinity eluate; how8 p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).
6
ISOLATION/CHARACTERIZATION
OF Ca-BINDING PROTEINS
[1]
ever, both recovery and nonspecific binding may vary with substituent level. When batches of melittin-Sepharose of different substituent concentrations (0.6 to I0 mg/ml gel) were compared, the apparent capacity for and recovery of CaM was directly proportional to the amount of immobilized melittin (Fig. 2). In the experiment shown, the slope of the line corresponded to a recovery of 0.7 mg CaM/mg melittin bound to the gel. Under optimum conditions (i.e., inclusion of 0.5 M NaCI in the elution buffer), the value increased to 0.8-0.9 mg CaM/mg bound melittin. These data indicate that melittin is immobilized to the beads in its monomeric form and that over this range of ligand density CaM can interact and be eluted without obvious problems such as steric hindrance or diminished recovery. The preferred ligand density for most applications would appear to be 5-10 mg melittin/ml gel, since this would permit both batchwise adsorption from extracts (see Method 1, below) as well as concentration of partially purified CaM. Because of its well-defined structure and properties, melittin provides an opportunity to describe, in quantitative terms, certain fundamental I
I
I
I
I
i
I
I
i
I
t~ iii I..J
tu
O~
E
4
2
2 6 10 SUBSTITUENT CONCENTRATION (mg Melittin/ml gel) FIG. 2. Interaction of CaM with melittin-Sepharose of different substituent concentrations. Melittin-Sepharose was prepared at substituent concentrations of 0.6, 0.7, 1.4, 2.8, 4.5, 5.4, and 10 mg of melittin/mg of gel. Partially purified CaM was made 1 mM in CaC12 and applied to the differently substituted columns in amounts sufficient to saturate the capacity of each column. After washing with four bed volumes of buffer A (25 mM BES, pH 7.0, containing 5 mM MgC12, l mM CaCI2, I mM NAN3, and 250 mM NaCI), columns were eluted with three bed volumes of buffer A lacking Ca 2÷ and containing 2 mM EGTA and 10% glycerol (buffer B). Eluted protein was quantitated using purified CaM as standard and normalized to the equivalent of I ml bed volume of melittin-Sepharose. The slope of the line indicates the recovery of CaM per milligram of melittin immobilized on the gel.
[1]
7
MELITTIN--SEPHAROSE AFFINITY CHROMATOGRAPHY I
I
I
I
2
4:
2.0 -- 94K
-11
o
--67K
3
1.5
m ~
--43K
Z
--30K
t~
I v
g 2
1.0
3 ~"
--21K i --
10
20 30 40 FRACTION NUMBER
14K
50
FIG. 3. Purification of CaM by affinity chromatography on melittin-Sepharose. Partially purified CaM (47 mg in 52 ml) was adjusted to 1 mM CaCI2 and applied to a column (2.5 x 8 cm, 40 ml) of melittin-Sepharose (0.6 mg melittin/ml gel) equilibrated in buffer A. After washing with 100 ml of buffer A containing 0.5 M NaCI (final concentration) at a flow rate of 120 ml/hr, the column was eluted (flow rate, 60 ml/hr) with 100 ml of buffer B, this is indicated by the arrow. Fractions (5 ml) were collected and assayed for protein (©) or absorbance at 280 nm (0). SDS-Gel electrophoresis of the starting material (4/zg), material not retained on the column (2.2/zg), and the EGTA eluate (4/zg) are shown in the inset, lanes 1-3, respectively. Positions of molecular weight standards (Pharmacia) run on the same gel are indicated on the right (phosphorylase a, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 21 kDa; a-lactalbumin, 14.4 kDa). Reprinted by permission from Ref. 1.
properties of affinity chromatography. The degree of nonspecific binding and ease of elution were most easily assessed in a simple chromatography step, using a partially purified DEAE fraction from bovine brain (Fig. 3). When this fraction was applied to a column of melittin-Sepharose in the presence of Ca 2÷, CaM was selectively retained while contaminating protein and nucleotide passed through. Minor amounts of nucleotide may interact with positively charged groups on the melittin-Sepharose; however, these can be eliminated by a washing step with 0.5 M NaC1. After addition of 2 mM EGTA, CaM was eluted as a sharp peak, indicating efficient reversal of the Ca2+-dependent protein-peptide association. This purified fraction appeared homogeneous by sodium dodecyl sulfate (SDS)-gel electrophoresis (inset, lane 3) and its ultraviolet absorption
8
[1]
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS A
I
'
I
'
I
#,
I --
2.0 --
~
-"
~--~-
--
2.0
3 -,n
o m z 1.2
e.-I
~
~
25
0.4 25
50
75
0.4
100
% CAPACITY
I
I
2
I
I
I
6 10 mg PROTEIN APPLIED
//I 18
--94K --67K --43K --30K --21K --14K 1
2
3
4
5
6
7
8
9
1011STD
FIG. 4. (A) Determination of the capacity of melittin-Sepharose for CaM and its relationship to recovery. The indicated amounts of partially purified CaM were applied to columns (0.7 x 1.8 cm, 0.7 ml) of melittin-Sepharose (4.5 mg melittipJml gel) equilibrated in buffer A, after which columns were washed with four bed volumes of that buffer and eluted with three bed volumes of buffer B. The amount of protein not retained was subtracted from that applied; the difference was the amount bound to the column (e). The amount of the protein in the eluate (O) was determined using bovine serum albumin as standard. Since all CaM applied was bound up to 4 mg of applied protein, the dashed line represents the amount of
[1]
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
9
spectrum showed essentially complete removal of nucleotide present in the starting material. In the preceding example, the amount of crude material containing CaM was calculated to just saturate the capacity of the melittinSepharose matrix; under these conditions, recovery of CaM (-80%), as well as its purity, was high. However, one potential pitfall of affinity chromatography relates to its properties under conditions of partial saturation; i.e., does recovery and/or specificity dramatically change when ligand sites are incompletely filled? To examine this question, increasing amounts of starting material were chromatographed on identical columns of melittin-Sepharose (Fig. 4). By comparing the total amount of protein bound (closed circles) with that eluted (open circles), it was evident that the percentage recovery increased as the column capacity became saturated, reaching a "limit" value for recovery of 80% (see inset, Fig. 4A). However, at 25% saturation of the capacity, recoveries were less than 50%. As shown in Fig. 4B, some "nonspecific" contaminants were also eluted when the column was only partially saturated, although these were effectively displaced by CaM at higher loads. The binding capacity of the matrix for CaM (see dashed line, Fig. 4A) was - 1 mg CaM/mg of bound melittin, corresponding to -16-20% of the available melittin sites. Since this same "capacity" was observed even at low substituent concentrations, it suggests that only particular orientations of bound melittin (one of five, on the average) are capable of high affinity associations. As there are four potentially reactive sites on melittin, this would imply that immobilization at one or two specific sites allows the high affinity association with CaM. Another Ca2÷-binding protein, S-I00 protein, which is present in brain, also was retained on melittin-Sepharose, albeit with lower affinity. Although S-100 (which migrates as a diffuse band near the dye front) appeared to be retained selectively at subsaturating loads (see lanes 5 and 7, Fig. 4B), it was displaced by CaM as column capacity was saturated (lanes 9 and 11, Fig. 4B). Thus, while both Ca2÷-binding proteins bind
CaM bound at lower amounts of applied protein; the shaded area represents the small amount of non-CaM protein adsorbed at these subsaturating levels. Inset: The percentage of bound CaM recovered in the eluate (ordinate) is plotted as a function of the percentage of column capacity (2 mg protein = 100% capacity). (B) SDS-gel electrophoresis of fractions from (A). Samples of unretained and eluted fractions (2-4 p.g) were precipitated with trichloroacetic acid (10%, w/v) and solubilized in buffer containing 1% SDS prior to electrophoresis. Lane 1, 4 p.g of starting material; lanes 2, 4, 6, 8, and I0, material not retained; lanes 3, 5, 7, 9, and I l, eluates from samples of 0.5, 1, 2, 4, and 8 mg of applied protein, respectively (see Fig. 4A). Reprinted by permission from Ref. 1.
10
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS
[1]
with high affinity, CaM can apparently compete for available ligand more effectively than S- 100. These data are consistent with basic principles of mass action law. In the absence of sufficient competing high-affinity interactions, lower affinity, "nonspecific" associations occur with bound ligand; these can be retained on the matrix and eluted. The quantitative relationship between column saturation and recovery is more difficult to explain. The lower recovery, which presumably reflects protein denaturation on the column, might be due to the low protein concentration of the eluate and/or rebinding to available ligand sites in advance of the elution front, although this explanation is speculative. In any event, saturation of column capacity is an important consideration, both to increase recovery and to minimize binding of weaker interacting species. One final element in characterizing the affinity matrix concerns its long-term stability and capacity. We have used the matrix six times for large-scale purification of CaM (see Method 2, below) and have not observed major changes in its binding capacity or recovery; also, the elution of nonspecific contaminants decreases after its first or second use, suggesting a reduction in available low-affinity sites. When using tissue extracts (see Method 1, below), the apparent capacity sometimes appeared to decrease with repeated usage; this may be due to proteases present in curde material which could degrade bound melittin. Since melittin contains trypsin-sensitive sites, it is quite important to include protease inhibitors in the initial batchwise adsorption step. Routine regeneration of the melittin-Sepharos e with "denaturing" buffers (see regeneration buffer above) also appeared to increase the stability of the matrix and reproducibility of the method.
Purification Methods Using Melittin-Sepharose
Method 1: Rapid Purification of CaM Directly from Crude Extracts Using Melittin-Sepharose/Organomercurical Agarose Chromatography Materials 100 g frozen tissue (e.g., bovine testis, brain, liver, heart, spleen) 16 ml melittin-Sepharose (5 mg melittin/Sepharose) 10 ml organomercurial agarose (Affi-Gel 501, Bio-Rad) 500 ml homogenization buffer [25 mM BES, pH 7.0/2.5 mM MgCIJ0.5 mM EGTA, 125 mM NaCI, plus 10/~g/ml soybean trypsin inhibitor (Worthington)]
[1]
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
I1
10 ml 250x protease inhibitor stock [0.5 g phenylmethylsulfonyl fluoride (PMSF), 10 mg pepstatin A, both from Sigma, dissolved in 20 ml dry methanol] 5 ml 100× antifoam A stock [antifoam A emulsion (Sigma) diluted 5fold in water] 100 ml buffer A (see legend to Fig. 2) 50 ml buffer A containing 0.5 M NaCI I00 ml buffer B (see legend to Fig. 2)
Procedure Step 1: Tissue Homogenization and Centrifugation of Extract. Frozen tissue which has been partially thawed is thinly sliced and added to 400 ml homogenization buffer containing appropriate dilutions of protease inhibitots (PMSF, pepstatin A) and antifoam emulsion (the latter prevents foaming during homogenization). The mixture is homogenized in a Waring blender three times at low speed for 10-15 sec. The homogenate is centrifuged at 25,000 g for 30 min, after which the supernatant is filtered through cheesecloth overlaid with a layer of glass wool. The volume of supernatant should be 380 - 20 ml. Step 2: Batchwise Adsorption to Melittin-Sepharose and Washing Steps. The supernatant is made 1 mM with Ca 2+ (4 ml of 0.1 M CaC12) and added to 16 ml of melittin-Sepharose in a container equipped for mixing (gentle stirring with a magnetic stir bar or end-over-end mixing in an appropriate flask). The suspension is mixed at 4 ° for a minimum of 2 hr, after which the gel is collected by brief centrifugation or allowed to settle by gravity. After separation from the bulk of the supernatant, the affinity gel is transferred to a column (2.5 cm diameter) with a minimum amount of washing buffer (buffer A) and packed. The melittin-Sepharose is then washed successively with 60 ml buffer A and 30 ml buffer A containing 0.5 M NaCI. Step 3: Elution of Melittin-Sepharose/Organomercurial Agarose. Following the washing steps, 10 ml of elution buffer (buffer B) is added to the column and the effluent discarded. After 30 min, the gel is eluted with 50 ml buffer B directly onto a column of organomercurial agarose (10 ml bed volume) positioned beneath the melittin-Sepharose column. After this, the elution is continued with an additional 15 ml buffer B. The fraction which passes through the organomercurial agarose (65 ml) should contain essentially only CaM; this can be concentrated by ultrafiltration to 20 ml. If desired, it may be applied to a second melittin-Sepharose column after addition of Ca 2+ to 3 mM (see Notes, below). Notes. Step 2: Collection of melittin-Sepharose is facilitated by centrifugation (2500 g, 5 min) in a swinging bucket rotor, followed by decant-
12
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS
i!i~iii!~!~ ~,i
[1]
....
~!~i! i~i,~~ii~~!!i i!ii~
-
94K
-
67K
-43K
-
30K
-20K
14.4K
i:
!ili :~
1
2
3
4
FIG. 5. Single-step purification of CaM from bovine testis supernatant. Thirty grams of bovine testis were homogenized in four volumes of 25 mM BES, pH 7.0, containing 100 mM NaC1, 2.5 MgC12, 0.5 mM EGTA and PMSF (75/.~g/ml). After centrifugation (20,000 g, 30 min), soybean trypsin inhibitor (20/zg/ml) was added to the supernatant (-100 ml), which was made 2 mM with CaCI2 and stirred with 6 ml of melittin-Sepharose (-4.5 mg melittin/ml gel) for 2 hr. The gel was collected by brief centrifugation, washed with 10 ml buffer A and
[1]
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
13
ing of supernatant. If the volume of the remaining slurry is still too great, a second brief centrifugation (i.e., in a 50-ml plastic centrifuge tube) can be done. Step 3: After use of the tandem affinity columns, the eluate containing CaM may appear homogeneous on SDS gels. However, the ultraviolet spectrum often shows additional absorbance at 260 nm, presumably due to residual nucleotide. It is therefore convenient to remove this absorbance and concentrate the CaM by a second chromatography step on a small (3-ml) column of melittin-Sepharose (10 mg melittin/ml). The procedure described above provides a rapid and general method for purification of CaM from a variety of tissues or extracts (e.g., bacterial lysates). Since CaM contains no free sulfhydryl groups, the use of the organomercurial columns selectively removes "nonspecific" protein contaminants which bind melittin-Sepharose as well as thiol-containing proteins exhibiting higher affinity interactions such as S-100 protein. Nonspecific interactions can be greatly diminished by "overloading" the melittin-Sepharose (i.e., increasing the ratio of tissue to affinity gel); indeed, a single-step purification from testis supernatant, without organomercurial agarose, was achieved by just saturating the binding capacity of a gel which had been used repeatedly (Fig. 5). Under these conditions, negligible nonspecific contaminants were observed in the final eluate, with a yield of 28 mg CAM/100 g testis. For many tissues, however, this may not be feasible due to the much lower concentration of CaM per volume of extract; in such cases, reasonable purity can still be obtained by using the tandem affinity column approach. A comparison of several bovine tissues using this protocol illustrates this point (Fig. 6). Although melittin-Sepharose chromatography, by itself, resulted in varying degrees of protein contaminants (especially in tissues with reduced concentrations of extractable CaM, such as heart), chromatography on organomercurial agarose removed the vast majority of these. Note the complete removal of S-100 protein present in the melittin-Sepharose eluate from brain extract (the S-I00 protein is the broad low-molecular-weight band in lane 1). Recoveries of CaM (based on addition of radiolabeled CaM to tissue supernatant) were routinely greater than 65% of that in the starting material. Similar results have also been observed with other bovine tis-
centrifuged a second time, and poured into a column (diameter 1.5 cm). The gel was washed with four bed volumes of buffer A containing 0.5 M NaC1 followed by 0.6 bed volumes of buffer B and, after 30 min, eluted with three bed volumes of buffer B (yield - 8 mg CAM); SDS-gel electrophoresis of supernatant, 6/zg (lane l), wash fraction, 2 gg (lane 2), melittinSepharose eluate, 4 ttg (lane 3), and protein standards (lane 4) as in Fig. 3. Reprinted by permission from Ref. I.
14
ISOLATION/CHARACTERIZATION
OF Ca-BINDING PROTEINS
[1]
Mr (kDa) --94 --67 --43 --30 --21 --14.5
1
2
(Brain)
3
4
(Heart)
5
6
(Liver)
7
8 Std.
(Testis)
Fro. 6. Purification of CaM from bovine tissues using melittin-Sepharose/organomercurial agarose chromatography. Tissue supernatants from bovine brain, heart, liver, and testis were prepared as described under method 1. After adsorption and elution of melittinSepharose, a portion of the eluate was removed prior to application to organomercurial agarose. Samples of melittin-Sepharose eluate (20/~1) and organomercurial agarose passthrough (40 p,1), respectively, were precipitated with trichloroacetic acid prior to SDS-gel electrophoresis. Brain (lanes 1, 2), heart (lanes 3, 4), liver (lanes 5, 6), testis (lanes 7, 8), and protein standards (lane 9). The yields of CAM/100 g tissue in this experiment were 15.5, 0.5, 10, 22 mg, respectively. sues (e.g., s p l e e n ) a n d n o n b o v i n e tissues (e.g., t u r k e y gizzard), suggesting the usefulness o f this a p p r o a c h in screening and preliminary purification f r o m o t h e r s o u r c e s .
Method 2: Large-Scale Procedure for Simultaneous Purification of CaM and S-100 Protein from Brain Materials 4 kg brain tissue (e.g., bovine, porcine, ovine) 120 ml m e l i t t i n - S e p h a r o s e (I0 mg melittin/ml Sepharose)
[1]
MELITTIN--SEPHAROSE AFFINITY CHROMATOGRAPHY
15
320 ml organomercurial agarose (Affi-Gel 501, Bio-Rad) 1.6 liters QAE-Sephadex A-25 20 liters homogenization buffer (as in Method 1, except lacking soybean trypsin inhibitor and including freshly deionized 6 M urea) 160 ml 100x antifoam A stock (as in Method 1) 20 ml 250x protease inhibitor stock (as in Method I) 1.2 liters buffer A (as in Method 1) 600 ml buffer A containing 0.5 M NaCI 1.2 liters buffer B (as in Method 1) 3 liters buffer C (homogenization buffer lacking 6 M urea) 6 liters buffer C containing 0.5 M NaCI
Procedure Step 1: Tissue Homogenization and Centrifugation of Extract. Tissue is homogenized in 16 liters of urea-containing homogenization buffer containing antifoam; this is most conveniently done in a large (6-liter capacity) homogenizer. The homogenate is centrifuged at low speed (e.g., 10 min at 3500 g in a swinging bucket rotor) or at medium flow rate in a continuous flow centrifuge (e.g., Sharpies) to remove gelatinous sediment. The supernatant at this point is extremely turbid; however, this does not interfere with subsequent steps. Step 2: Batchwise Chromatography with Anion-Exchange Media~ To the supernatant, 1.6 liters of QAE-Sephadex A-25 (thoroughly equilibrated in urea-containing homogenization buffer) is added with efficient stirring. The suspension is vigorously mixed for at least 2-4 hr, after which the gel is allowed to settle overnight. The bulk of the supernatant then can be removed by carefully siphoning to a point just above the level of the settled gel. The gel can then be slurried with a minimum volume of homogenization buffer and transferred to a porous support such as a sintered glass funnel. Alternatively, the gel can be collected by centrifugation (e.g., in a Sharples centrifuge) immediately after mixing and packed on a support. After the gel has packed, it is washed with 2 liters of ureacontaining buffer to remove any trapped supernatant, followed by washing with 3 liters of buffer C. The gel is then eluted with 5 liters of buffer C containing 0.5 M NaC1 and concentrated to 800 ml by high-volume ultrafiltration (120 ml/min; Pellicon cassette system, PTGC membrane, Millipore Corp.).
Step 3: Organomercurial Agarose/Melittin-Sepharose Chromatography Steps. The concentrated anion-exchange eluate is made 2 mM in calcium (16 ml of 0.1 M CaC1) and a 200-ml portion of this is applied to a bed of organomercurial agarose (320 ml on a 600-ml sintered glass funnel) which was previously equilibrated in buffer A. The effluent from this step is passed directly through a column of melittin-Sepharose (120 ml in a
16
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS
[1]
4-cm-diameter column) equilibrated in buffer A. Successive 200-ml additions are made at intervals which allow effluent to pass completely through the melittin-Sepharose. After sample application is complete, the Affi-Gel 501 is washed twice with 200 ml portions of buffer A, again directly onto the column of melittin-Sepharose. The two affinity gels are then separated and each washed with two bed volumes of buffer A containing 0.5 M NaCI. To the washed melittin-Sepharose 75 ml of buffer B are added and the effluent discarded. After 30 min, this gel is eluted with 360 ml of buffer B to yield essentially homogeneous CaM (1.0-1.4 g). The melittin-Sepharose is exhaustively regenerated with low pH urea buffer (see preparation of affinity matrix) followed by equilibration in buffer A. To the washed organomercurial gel (which contains bound S-100 protein) 200 ml of buffer A containing 10 mM dithiothreitol is added and the effluent discarded. The gel is then eluted with four successive 200-ml additions of thiol-containing buffer directly onto the equilibrated melittinSepharose gel as described above. The two affinity gels are then separated and the melittin-Sepharose washed with 240 ml of buffer A containing 0.5 M NaCI. To the washed melittin-Sepharose 75 ml of buffer B is added and the effluent discarded. After 30 min, the gel is eluted with 360 ml of buffer B to yield essentially homogeneous S-100 protein (0.8-1.0 g). Notes. Step 1: Urea used in the homogenization buffer should be deionized just before use to minimize ionic impurities (e.g., cyanates); this can be done by rapidly passing an 8 M urea solution over a mixed bed resin such as Rexyn 1-300 (Fisher). Step 2: It is crucial to wash the packed QAE gel with at least 1.2 bed volumes of urea-containing buffer before proceeding with buffer lacking urea; if this is not done, urea-soluble components in the trapped supernatant precipitate and stop flow through the gel. Step 3: If only CaM is desired, melittin-Sepharose chromatography can be carried out directly, omitting the organomercurial step; the eluate should be passed over a small column (60 ml) of Affi-Gel 501 to remove any contaminating S-100 protein. Although melittin-Sepharose chromatography of supernatant fractions (Method 1) gives excellent results, the use of the urea extraction of brain tissue, followed by a batchwise ion-exchange step prior to melittinSepharose, is preferable for large-scale procedures. Because only rapid, low-speed centrifugation of the homogenate is required, larger amounts of tissue can be processed with relative ease in conventional centrifuges with production of very little insoluble material. Furthermore, homogenization in 6 M urea solubilizes a large amount of brain CaM (40%) which is otherwise difficult to extract from the particulate fraction. The subsequent use of QAE-Sephadex A-25 provides, in addition to an ion-ex-
[1]
17
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
! J Q-I
~10.0
;, 3.2
o~
E i
z
iii I0 E:
"
2.4
6.0 B
2.0
¢0 0
÷= ~ Z
1.6
m
,o.8
g -I
3 20
30
40
50
60
70
FRACTION NUMBER Fro. 7. Gel filtration of QAE-Sephadex A-25 eluate. The anion-exchange eluate (30 ml, 45 mg protein/ml), prepared as described for method 2, was chromatographed on a column (94 × 5 cm) of Ultragel AcA 54 (LKB) at a flow rate of 80 ml/hr. Fractions of 20 ml were collected and monitored for protein ( - - A - - ) and A280 ( - - A - - ) . Portions (-10/zg) of the starting material (Q-l) and selected fractions, as indicated by the bent lines, were analyzed by SDS-gel electrophoresis (inset). The two proteins in the major peak of protein are CaM (upper band) and S-100 protein (lower band).
change step, selective binding of smaller proteins and nucleotides; the eluate from this step shows essentially only CaM and S-100 protein with minor amounts of other higher molecular weight proteins (Fig. 7). As noted earlier, when CaM and S-100 protein are both present in the fraction applied to melittin-Sepharose, CaM efficiently displaces S-100 protein, resulting in a small amount of the latter protein in the eluate. Since SI00 protein contains free sulfhydryl groups, while CaM does not, they can be separated quantitatively by using a small column of organomercurial agarose (Fig. 8). The yield of CaM when this procedure is used ranges from 270-350 mg/kg tissue and is the same from bovine, ovine, or porcine brain. One such preparation, from 6 kg tissue is shown in the table. If S100 protein is also desired, the use of the sulfhydryl adsorbent prior to melittin-Sepharose permits preparative isolation of both proteins in com-
18
ISOLATION/CHARACTERIZATION
OF Ca-BINDING PROTEINS
[1]
--94K --67K --43K --30K -21K -- 14K
1
2
3
4
STD
FIG. 8. Separation of CaM and S-100 protein usmg organomercunal agarose chromatography. For this experiment, CaM was prepared by method 2 through elution of the melittinSepharose (prior adsorption of S-100 protein on the sulfhydryl gel was omitted). A portion of the eluate (5 ml, 5 mg of protein) was applied to a column (0.7 x 3.6 cm, 1.5 ml) of organomercurial agarose (Affi-Gel 501, Bio-Rad). This column was washed with 1.2 bed volumes of buffer B, and the effluent was added to the fraction not retained. After washing with three bed volumes of buffer B, the column was eluted with three bed volumes of buffer B containing 10 mM dithiothreitol. Proteins were precipitated and solubilized for SDS-gel electrophoresis. Lane 1, 5/xl of starting material; lane 2, 7/zl of material not retained on organomercurial agarose (CaM); lane 3, 5/~1 of the wash fraction; lane 4, 5/~1 of the eluate (S-100 protein). Coomassie blue-stained material in the region of 60-70 kDa is a gel artifact, since it was found in lanes which contained no protein. Reprinted by permission from Ref. 1.
[2]
CALCIMEDINS
19
TABLE I LARGE-SCALE PURIFICATION OF CALMODUL1N AND S-100 PROTEIN a
Preparation 1 (bovine brain)
Step Homogenate QAE-Sephadex Melittin-Sepharose Eluate 1 (CAM) Melittin-Sepharose Eluate 2 (S-100)
Preparation 2 (ovine brain)
Volume (ml)
Protein (mg)
Yield (mg/kg tissue)
Volume (ml)
Protein (rag)
Yield (mg/kg tissue)
27,600 1,040 540
-2,920 1,920 b
l -320
17,800 800 360
-2,180 1,350 b
--338
--
--
--
1,080
270
360
a The purification procedure was essentially as outlined under method 2 starting with either 6 kg (preparation 1) or 4 kg (preparation 2) of brain tissue. In preparation 1, the large-scale organomercurial agarose step was omitted and the QAE-Sephadex eluate applied directly to the melittin-Sepharose; the eluate from this latter step was depleted of a small amount of S-100 by passage over a column of Affi-Gel 501 (see Fig. 8). Estimates of protein were made using the G-250 dye-binding assay (Bio-Rad) with bovine serum albumin as standard. b Protein for calmodulin determined using purified calmodulin as standard.
parable yield (Table I). Because of the speed, reproducibility, and scaleup potential of this procedure, it should prove useful for investigators involved in physical and structural studies of the two proteins where large quantities of protein are necessary.
[2] C a l c i m e d i n s : P u r i f i c a t i o n a n d P r o d u c t i o n o f A n t i b o d i e s B y J . K . M A T H E W , R . R . SCULLY, V . L . S M I T H , E . BERNICKER, a n d J. R. DEDMAN
Physiological responses in excitable cells are often preceded by an elevation of free calcium. ~This intracellular "calcium signal" is mediated by high-affinity, metal-selective protein receptors. 2 The first such protein mediator identified was the troponin complex of striated muscle) One I H. Rasmussen, in "Calcium and Cell Function" (W. Y. Cheung, ed.), Vol. 4, p. 1. Academic Press, New York, 1983. 2 A. R. Means and J. R. Dedman, Nature (London) 285, 73 (1980). 3 p. B. Moore and J. R. Dedman, J. Biol. Chem. 257, 9663 (1982).
METHODS IN ENZYMOLOGY, VOL. 139
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
3
[1] T h e U s e o f M e l i t t i n - S e p h a r o s e C h r o m a t o g r a p h y for Gram-Preparative Purification of Calmodulin By RANDALL L. KINCAID
Introduction
The successful application of affinity chromatography relies on the specific retention of macromolecules on a matrix of immobilized ligand. Ideally, the properties of the matrix should meet several criteria. First, the interaction should be of high affinity (Kd - 10 -7 M ) to enable rigorous washing of the gel without desorption of bound material. Second, the association of bound proteins with ligand should be readily reversed by addition of an eluting agent which either competes for ligand (substrate competition) or which greatly reduces the affinity of the ligand-macromolecule complex. Last, the properties of the matrix should be well defined, permitting quantitative analysis, and stable to repeated usage and regeneration procedures. With these considerations in mind, we prepared an affinity matrix of immobilized melittin for purification of calmodulin (CAM). z Melittin, the major component of bee venom, is a 26 amino acid peptide which is amphipathic; it contains a long stretch of hydrophobic residues, a single aromatic residue (tryptophan, position 19) and several positively charged side chains clustered near the carboxyl terminus (Fig. 1). This peptide, which aggregates into tetramers at concentrations of 0.2 mg/ml and greater, 2 is soluble in both aqueous and lipid environments; as a consequence, melittin may have a general biological role as a surfactant. 3 Interestingly, several laboratories noted that mdittin inhibited cyclic nucleotide phosphodiesterase 4,5 and reported that inhibition was dependent on Ca 2+ and competitive with CaM. Later, physical studies documented direct stoichiometric interaction of melittin with CaM by noting changes in its fluorescence anisotropy 6,7 and by quantifying complexes formed during electrophoresis and gel filtration chromatography. 5 In I R. L. Kincaid and C. C. Coulson, Biochem. Biophys. Res. Commun. 133, 256 (1985). 2 T. C. Terwilliger and D. Eisenberg, J. Biol. Chem. 257, 6010 (1982). 3 E. Kn6ppel, D. Eisenberg, and W. Wickner, Biochemistry 18, 4177 (1979). 4 M. S. Barnette, R. Daly, and B. Weiss, Biochem. Pharmacol. 32, 2929 (1983). 5 M. Comte, Y. Maulet, and J. A. Cox, Biochem. J. 209, 269 (1983). 6 y . Maulet and J. A. Cox, Biochemistry 22, 5680 (1983). 7 D. A. Malencick and S. R. Anderson, Biochemistry 23, 2420 (1984).
METHODS IN ENZYMOLOGY, VOL. 139
4
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS
/ ~
NH 2
.
'.p
[1]
/J-~
A
'"
)
FIG. l. Schematicrepresentationof the aminoacid sequenceof melittin. The numbered circles indicate successiveaminoacids beginningwith the aminoterminus. The projections represent the carbon bonds in the side chains of each amino acid; positive charges are indicated for the lysineresidues at positions 7, 20, and 22 and for arginine at positions 21 and 23. those studies and from work in our own laboratory (R. Kincaid, unpublished data), the affinity of melittin for CaM was estimated to be in the nanomolar range. Because of this high binding constant, as well as its well-defined properties and availability, it seemed an appropriate ligand for the Ca2+-dependent affinity chromatography of CaM and perhaps other Ca2÷-binding proteins. Our studies have shown that melittinSepharose is suitable for large-scale purification of CaM and S-I00 protein from brain and that, in some tissues, purification of CaM to an essentially homogeneous state can be accomplished in a single step. The present chapter outlines analytical and preparative details for use of this affinity matrix. Preparation of Melittin-Sepharose (10 mg Melittin/ml Gel)
Materials 15 g CNBr-activated Sepharose 4B (Pharmacia) 4 liters HCI, 1 mM 0.2 liter coupling buffer (0.1 M sodium bicarbonate, pH 8.5, containing 0.5 M NaC1) 500 mg melittin, 10 mg/ml, dissolved in coupling buffer (Sigma or U.S. Biochemical Corp.) 75 ml 1 M ethanolamine- HCI, pH 8.0 0.2 liter low-pH urea regeneration buffer (0.1 M sodium acetate, pH 4.5, containing 6 M urea, I0 mM EDTA, and 0.5 M NaCI) 0.2 liter high-pH wash buffer (0.1 M sodium borate, pH 9.0, containing 0.5 M NaCI)
[1]
MELITTIN--SEPHAROSE AFFINITY CHROMATOGRAPHY
5
Procedure Step 1: Washing of CNBr-Sepharose 4B. The lyophilized CNBrSepharose 4B is swollen for 5 min in 100 ml 1 mM HC1 with gentle mixing. It is then transferred to a l-liter sintered glass funnel (coarse frit), resuspended in 400 ml 1 mM HCI, and filtered with suction to form a moist cake. The gel is suspended and filtered three more times, after which the gel cake (45-50 ml, bed volume) is transferred to a beaker and mixed with 25 ml i mM HC1. Step 2: Coupling of Melittin to CNBr-Sepharose 4B. To the washed gel, 50 ml of melittin (10 mg/ml) dissolved in coupling buffer is added and mixed immediately. To this, 7.5 ml each of 1 M sodium bicarbonate, pH 8.5, and 5 M NaCl are added to make final concentrations of 0.1 and 0.5 M, respectively. The slurry is transferred to a 250-ml plastic tissue culture flask (Falcon) and mixed on a tilting rocker (e.g., Thermolyne clinical test tube mixer). Step 3: Blocking of Excess Reactive Groups on Melittin-Sepharose. After 2 hr at room temperature, the suspension is suctioned on a 350-ml sintered glass funnel and the moist cake washed with 75 ml coupling buffer. The gel is transferred to a beaker and 25 ml coupling buffer is added. To this slurry 75 ml of 1 M ethanolamine- HCI, pH 8.0, is added and the suspension mixed for 2 hr as in step 2. Step 4: Washing and Storage of Melittin-Sepharose. After blocking, the gel is transfered to a 350-ml sintered glass funnel and suctioned. The gel is washed with 100 ml of low pH urea regeneration buffer, followed by 100 ml high pH wash buffer; this washing cycle is repeated. The gel is suspended in 50 ml of 20 mM 2-[bis-2[hydroxyethyl)amino]ethanesulfonic acid (BES) (Calbiochem), pH 7.2, containing 100 mM NaCl and 0.02% Thimerosal (Sigma) and stored at 4 °. Notes on Procedure. Step 1: Sepharose can also be activated using 150 mg CNBr/ml Sepharose 4B, at alkaline pH, as described, a Step 2: A substituent density of 5 mg melittin/ml gel is achieved by adding one-half the amount (i.e., 25 ml) of melittin solution. Step 3: The use of a low-pH urea-containing buffer is important to remove some colored, hydrophobic impurities. Optimization and Quantitative Properties of the Affinity Matrix An important step in preparation of an affinity matrix is to establish the optimum relationship between substituent concentration on the gel and binding capacity/recovery. Clearly, it would be best to achieve a high enough binding capacity to ensure a concentrated affinity eluate; how8 p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).
6
ISOLATION/CHARACTERIZATION
OF Ca-BINDING PROTEINS
[1]
ever, both recovery and nonspecific binding may vary with substituent level. When batches of melittin-Sepharose of different substituent concentrations (0.6 to I0 mg/ml gel) were compared, the apparent capacity for and recovery of CaM was directly proportional to the amount of immobilized melittin (Fig. 2). In the experiment shown, the slope of the line corresponded to a recovery of 0.7 mg CaM/mg melittin bound to the gel. Under optimum conditions (i.e., inclusion of 0.5 M NaCI in the elution buffer), the value increased to 0.8-0.9 mg CaM/mg bound melittin. These data indicate that melittin is immobilized to the beads in its monomeric form and that over this range of ligand density CaM can interact and be eluted without obvious problems such as steric hindrance or diminished recovery. The preferred ligand density for most applications would appear to be 5-10 mg melittin/ml gel, since this would permit both batchwise adsorption from extracts (see Method 1, below) as well as concentration of partially purified CaM. Because of its well-defined structure and properties, melittin provides an opportunity to describe, in quantitative terms, certain fundamental I
I
I
I
I
i
I
I
i
I
t~ iii I..J
tu
O~
E
4
2
2 6 10 SUBSTITUENT CONCENTRATION (mg Melittin/ml gel) FIG. 2. Interaction of CaM with melittin-Sepharose of different substituent concentrations. Melittin-Sepharose was prepared at substituent concentrations of 0.6, 0.7, 1.4, 2.8, 4.5, 5.4, and 10 mg of melittin/mg of gel. Partially purified CaM was made 1 mM in CaC12 and applied to the differently substituted columns in amounts sufficient to saturate the capacity of each column. After washing with four bed volumes of buffer A (25 mM BES, pH 7.0, containing 5 mM MgC12, l mM CaCI2, I mM NAN3, and 250 mM NaCI), columns were eluted with three bed volumes of buffer A lacking Ca 2÷ and containing 2 mM EGTA and 10% glycerol (buffer B). Eluted protein was quantitated using purified CaM as standard and normalized to the equivalent of I ml bed volume of melittin-Sepharose. The slope of the line indicates the recovery of CaM per milligram of melittin immobilized on the gel.
[1]
7
MELITTIN--SEPHAROSE AFFINITY CHROMATOGRAPHY I
I
I
I
2
4:
2.0 -- 94K
-11
o
--67K
3
1.5
m ~
--43K
Z
--30K
t~
I v
g 2
1.0
3 ~"
--21K i --
10
20 30 40 FRACTION NUMBER
14K
50
FIG. 3. Purification of CaM by affinity chromatography on melittin-Sepharose. Partially purified CaM (47 mg in 52 ml) was adjusted to 1 mM CaCI2 and applied to a column (2.5 x 8 cm, 40 ml) of melittin-Sepharose (0.6 mg melittin/ml gel) equilibrated in buffer A. After washing with 100 ml of buffer A containing 0.5 M NaCI (final concentration) at a flow rate of 120 ml/hr, the column was eluted (flow rate, 60 ml/hr) with 100 ml of buffer B, this is indicated by the arrow. Fractions (5 ml) were collected and assayed for protein (©) or absorbance at 280 nm (0). SDS-Gel electrophoresis of the starting material (4/zg), material not retained on the column (2.2/zg), and the EGTA eluate (4/zg) are shown in the inset, lanes 1-3, respectively. Positions of molecular weight standards (Pharmacia) run on the same gel are indicated on the right (phosphorylase a, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 21 kDa; a-lactalbumin, 14.4 kDa). Reprinted by permission from Ref. 1.
properties of affinity chromatography. The degree of nonspecific binding and ease of elution were most easily assessed in a simple chromatography step, using a partially purified DEAE fraction from bovine brain (Fig. 3). When this fraction was applied to a column of melittin-Sepharose in the presence of Ca 2÷, CaM was selectively retained while contaminating protein and nucleotide passed through. Minor amounts of nucleotide may interact with positively charged groups on the melittin-Sepharose; however, these can be eliminated by a washing step with 0.5 M NaC1. After addition of 2 mM EGTA, CaM was eluted as a sharp peak, indicating efficient reversal of the Ca2+-dependent protein-peptide association. This purified fraction appeared homogeneous by sodium dodecyl sulfate (SDS)-gel electrophoresis (inset, lane 3) and its ultraviolet absorption
8
[1]
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS A
I
'
I
'
I
#,
I --
2.0 --
~
-"
~--~-
--
2.0
3 -,n
o m z 1.2
e.-I
~
~
25
0.4 25
50
75
0.4
100
% CAPACITY
I
I
2
I
I
I
6 10 mg PROTEIN APPLIED
//I 18
--94K --67K --43K --30K --21K --14K 1
2
3
4
5
6
7
8
9
1011STD
FIG. 4. (A) Determination of the capacity of melittin-Sepharose for CaM and its relationship to recovery. The indicated amounts of partially purified CaM were applied to columns (0.7 x 1.8 cm, 0.7 ml) of melittin-Sepharose (4.5 mg melittipJml gel) equilibrated in buffer A, after which columns were washed with four bed volumes of that buffer and eluted with three bed volumes of buffer B. The amount of protein not retained was subtracted from that applied; the difference was the amount bound to the column (e). The amount of the protein in the eluate (O) was determined using bovine serum albumin as standard. Since all CaM applied was bound up to 4 mg of applied protein, the dashed line represents the amount of
[1]
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
9
spectrum showed essentially complete removal of nucleotide present in the starting material. In the preceding example, the amount of crude material containing CaM was calculated to just saturate the capacity of the melittinSepharose matrix; under these conditions, recovery of CaM (-80%), as well as its purity, was high. However, one potential pitfall of affinity chromatography relates to its properties under conditions of partial saturation; i.e., does recovery and/or specificity dramatically change when ligand sites are incompletely filled? To examine this question, increasing amounts of starting material were chromatographed on identical columns of melittin-Sepharose (Fig. 4). By comparing the total amount of protein bound (closed circles) with that eluted (open circles), it was evident that the percentage recovery increased as the column capacity became saturated, reaching a "limit" value for recovery of 80% (see inset, Fig. 4A). However, at 25% saturation of the capacity, recoveries were less than 50%. As shown in Fig. 4B, some "nonspecific" contaminants were also eluted when the column was only partially saturated, although these were effectively displaced by CaM at higher loads. The binding capacity of the matrix for CaM (see dashed line, Fig. 4A) was - 1 mg CaM/mg of bound melittin, corresponding to -16-20% of the available melittin sites. Since this same "capacity" was observed even at low substituent concentrations, it suggests that only particular orientations of bound melittin (one of five, on the average) are capable of high affinity associations. As there are four potentially reactive sites on melittin, this would imply that immobilization at one or two specific sites allows the high affinity association with CaM. Another Ca2÷-binding protein, S-I00 protein, which is present in brain, also was retained on melittin-Sepharose, albeit with lower affinity. Although S-100 (which migrates as a diffuse band near the dye front) appeared to be retained selectively at subsaturating loads (see lanes 5 and 7, Fig. 4B), it was displaced by CaM as column capacity was saturated (lanes 9 and 11, Fig. 4B). Thus, while both Ca2÷-binding proteins bind
CaM bound at lower amounts of applied protein; the shaded area represents the small amount of non-CaM protein adsorbed at these subsaturating levels. Inset: The percentage of bound CaM recovered in the eluate (ordinate) is plotted as a function of the percentage of column capacity (2 mg protein = 100% capacity). (B) SDS-gel electrophoresis of fractions from (A). Samples of unretained and eluted fractions (2-4 p.g) were precipitated with trichloroacetic acid (10%, w/v) and solubilized in buffer containing 1% SDS prior to electrophoresis. Lane 1, 4 p.g of starting material; lanes 2, 4, 6, 8, and I0, material not retained; lanes 3, 5, 7, 9, and I l, eluates from samples of 0.5, 1, 2, 4, and 8 mg of applied protein, respectively (see Fig. 4A). Reprinted by permission from Ref. 1.
10
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS
[1]
with high affinity, CaM can apparently compete for available ligand more effectively than S- 100. These data are consistent with basic principles of mass action law. In the absence of sufficient competing high-affinity interactions, lower affinity, "nonspecific" associations occur with bound ligand; these can be retained on the matrix and eluted. The quantitative relationship between column saturation and recovery is more difficult to explain. The lower recovery, which presumably reflects protein denaturation on the column, might be due to the low protein concentration of the eluate and/or rebinding to available ligand sites in advance of the elution front, although this explanation is speculative. In any event, saturation of column capacity is an important consideration, both to increase recovery and to minimize binding of weaker interacting species. One final element in characterizing the affinity matrix concerns its long-term stability and capacity. We have used the matrix six times for large-scale purification of CaM (see Method 2, below) and have not observed major changes in its binding capacity or recovery; also, the elution of nonspecific contaminants decreases after its first or second use, suggesting a reduction in available low-affinity sites. When using tissue extracts (see Method 1, below), the apparent capacity sometimes appeared to decrease with repeated usage; this may be due to proteases present in curde material which could degrade bound melittin. Since melittin contains trypsin-sensitive sites, it is quite important to include protease inhibitors in the initial batchwise adsorption step. Routine regeneration of the melittin-Sepharos e with "denaturing" buffers (see regeneration buffer above) also appeared to increase the stability of the matrix and reproducibility of the method.
Purification Methods Using Melittin-Sepharose
Method 1: Rapid Purification of CaM Directly from Crude Extracts Using Melittin-Sepharose/Organomercurical Agarose Chromatography Materials 100 g frozen tissue (e.g., bovine testis, brain, liver, heart, spleen) 16 ml melittin-Sepharose (5 mg melittin/Sepharose) 10 ml organomercurial agarose (Affi-Gel 501, Bio-Rad) 500 ml homogenization buffer [25 mM BES, pH 7.0/2.5 mM MgCIJ0.5 mM EGTA, 125 mM NaCI, plus 10/~g/ml soybean trypsin inhibitor (Worthington)]
[1]
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
I1
10 ml 250x protease inhibitor stock [0.5 g phenylmethylsulfonyl fluoride (PMSF), 10 mg pepstatin A, both from Sigma, dissolved in 20 ml dry methanol] 5 ml 100× antifoam A stock [antifoam A emulsion (Sigma) diluted 5fold in water] 100 ml buffer A (see legend to Fig. 2) 50 ml buffer A containing 0.5 M NaCI I00 ml buffer B (see legend to Fig. 2)
Procedure Step 1: Tissue Homogenization and Centrifugation of Extract. Frozen tissue which has been partially thawed is thinly sliced and added to 400 ml homogenization buffer containing appropriate dilutions of protease inhibitots (PMSF, pepstatin A) and antifoam emulsion (the latter prevents foaming during homogenization). The mixture is homogenized in a Waring blender three times at low speed for 10-15 sec. The homogenate is centrifuged at 25,000 g for 30 min, after which the supernatant is filtered through cheesecloth overlaid with a layer of glass wool. The volume of supernatant should be 380 - 20 ml. Step 2: Batchwise Adsorption to Melittin-Sepharose and Washing Steps. The supernatant is made 1 mM with Ca 2+ (4 ml of 0.1 M CaC12) and added to 16 ml of melittin-Sepharose in a container equipped for mixing (gentle stirring with a magnetic stir bar or end-over-end mixing in an appropriate flask). The suspension is mixed at 4 ° for a minimum of 2 hr, after which the gel is collected by brief centrifugation or allowed to settle by gravity. After separation from the bulk of the supernatant, the affinity gel is transferred to a column (2.5 cm diameter) with a minimum amount of washing buffer (buffer A) and packed. The melittin-Sepharose is then washed successively with 60 ml buffer A and 30 ml buffer A containing 0.5 M NaCI. Step 3: Elution of Melittin-Sepharose/Organomercurial Agarose. Following the washing steps, 10 ml of elution buffer (buffer B) is added to the column and the effluent discarded. After 30 min, the gel is eluted with 50 ml buffer B directly onto a column of organomercurial agarose (10 ml bed volume) positioned beneath the melittin-Sepharose column. After this, the elution is continued with an additional 15 ml buffer B. The fraction which passes through the organomercurial agarose (65 ml) should contain essentially only CaM; this can be concentrated by ultrafiltration to 20 ml. If desired, it may be applied to a second melittin-Sepharose column after addition of Ca 2+ to 3 mM (see Notes, below). Notes. Step 2: Collection of melittin-Sepharose is facilitated by centrifugation (2500 g, 5 min) in a swinging bucket rotor, followed by decant-
12
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS
i!i~iii!~!~ ~,i
[1]
....
~!~i! i~i,~~ii~~!!i i!ii~
-
94K
-
67K
-43K
-
30K
-20K
14.4K
i:
!ili :~
1
2
3
4
FIG. 5. Single-step purification of CaM from bovine testis supernatant. Thirty grams of bovine testis were homogenized in four volumes of 25 mM BES, pH 7.0, containing 100 mM NaC1, 2.5 MgC12, 0.5 mM EGTA and PMSF (75/.~g/ml). After centrifugation (20,000 g, 30 min), soybean trypsin inhibitor (20/zg/ml) was added to the supernatant (-100 ml), which was made 2 mM with CaCI2 and stirred with 6 ml of melittin-Sepharose (-4.5 mg melittin/ml gel) for 2 hr. The gel was collected by brief centrifugation, washed with 10 ml buffer A and
[1]
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
13
ing of supernatant. If the volume of the remaining slurry is still too great, a second brief centrifugation (i.e., in a 50-ml plastic centrifuge tube) can be done. Step 3: After use of the tandem affinity columns, the eluate containing CaM may appear homogeneous on SDS gels. However, the ultraviolet spectrum often shows additional absorbance at 260 nm, presumably due to residual nucleotide. It is therefore convenient to remove this absorbance and concentrate the CaM by a second chromatography step on a small (3-ml) column of melittin-Sepharose (10 mg melittin/ml). The procedure described above provides a rapid and general method for purification of CaM from a variety of tissues or extracts (e.g., bacterial lysates). Since CaM contains no free sulfhydryl groups, the use of the organomercurial columns selectively removes "nonspecific" protein contaminants which bind melittin-Sepharose as well as thiol-containing proteins exhibiting higher affinity interactions such as S-100 protein. Nonspecific interactions can be greatly diminished by "overloading" the melittin-Sepharose (i.e., increasing the ratio of tissue to affinity gel); indeed, a single-step purification from testis supernatant, without organomercurial agarose, was achieved by just saturating the binding capacity of a gel which had been used repeatedly (Fig. 5). Under these conditions, negligible nonspecific contaminants were observed in the final eluate, with a yield of 28 mg CAM/100 g testis. For many tissues, however, this may not be feasible due to the much lower concentration of CaM per volume of extract; in such cases, reasonable purity can still be obtained by using the tandem affinity column approach. A comparison of several bovine tissues using this protocol illustrates this point (Fig. 6). Although melittin-Sepharose chromatography, by itself, resulted in varying degrees of protein contaminants (especially in tissues with reduced concentrations of extractable CaM, such as heart), chromatography on organomercurial agarose removed the vast majority of these. Note the complete removal of S-100 protein present in the melittin-Sepharose eluate from brain extract (the S-I00 protein is the broad low-molecular-weight band in lane 1). Recoveries of CaM (based on addition of radiolabeled CaM to tissue supernatant) were routinely greater than 65% of that in the starting material. Similar results have also been observed with other bovine tis-
centrifuged a second time, and poured into a column (diameter 1.5 cm). The gel was washed with four bed volumes of buffer A containing 0.5 M NaC1 followed by 0.6 bed volumes of buffer B and, after 30 min, eluted with three bed volumes of buffer B (yield - 8 mg CAM); SDS-gel electrophoresis of supernatant, 6/zg (lane l), wash fraction, 2 gg (lane 2), melittinSepharose eluate, 4 ttg (lane 3), and protein standards (lane 4) as in Fig. 3. Reprinted by permission from Ref. I.
14
ISOLATION/CHARACTERIZATION
OF Ca-BINDING PROTEINS
[1]
Mr (kDa) --94 --67 --43 --30 --21 --14.5
1
2
(Brain)
3
4
(Heart)
5
6
(Liver)
7
8 Std.
(Testis)
Fro. 6. Purification of CaM from bovine tissues using melittin-Sepharose/organomercurial agarose chromatography. Tissue supernatants from bovine brain, heart, liver, and testis were prepared as described under method 1. After adsorption and elution of melittinSepharose, a portion of the eluate was removed prior to application to organomercurial agarose. Samples of melittin-Sepharose eluate (20/~1) and organomercurial agarose passthrough (40 p,1), respectively, were precipitated with trichloroacetic acid prior to SDS-gel electrophoresis. Brain (lanes 1, 2), heart (lanes 3, 4), liver (lanes 5, 6), testis (lanes 7, 8), and protein standards (lane 9). The yields of CAM/100 g tissue in this experiment were 15.5, 0.5, 10, 22 mg, respectively. sues (e.g., s p l e e n ) a n d n o n b o v i n e tissues (e.g., t u r k e y gizzard), suggesting the usefulness o f this a p p r o a c h in screening and preliminary purification f r o m o t h e r s o u r c e s .
Method 2: Large-Scale Procedure for Simultaneous Purification of CaM and S-100 Protein from Brain Materials 4 kg brain tissue (e.g., bovine, porcine, ovine) 120 ml m e l i t t i n - S e p h a r o s e (I0 mg melittin/ml Sepharose)
[1]
MELITTIN--SEPHAROSE AFFINITY CHROMATOGRAPHY
15
320 ml organomercurial agarose (Affi-Gel 501, Bio-Rad) 1.6 liters QAE-Sephadex A-25 20 liters homogenization buffer (as in Method 1, except lacking soybean trypsin inhibitor and including freshly deionized 6 M urea) 160 ml 100x antifoam A stock (as in Method 1) 20 ml 250x protease inhibitor stock (as in Method I) 1.2 liters buffer A (as in Method 1) 600 ml buffer A containing 0.5 M NaCI 1.2 liters buffer B (as in Method 1) 3 liters buffer C (homogenization buffer lacking 6 M urea) 6 liters buffer C containing 0.5 M NaCI
Procedure Step 1: Tissue Homogenization and Centrifugation of Extract. Tissue is homogenized in 16 liters of urea-containing homogenization buffer containing antifoam; this is most conveniently done in a large (6-liter capacity) homogenizer. The homogenate is centrifuged at low speed (e.g., 10 min at 3500 g in a swinging bucket rotor) or at medium flow rate in a continuous flow centrifuge (e.g., Sharpies) to remove gelatinous sediment. The supernatant at this point is extremely turbid; however, this does not interfere with subsequent steps. Step 2: Batchwise Chromatography with Anion-Exchange Media~ To the supernatant, 1.6 liters of QAE-Sephadex A-25 (thoroughly equilibrated in urea-containing homogenization buffer) is added with efficient stirring. The suspension is vigorously mixed for at least 2-4 hr, after which the gel is allowed to settle overnight. The bulk of the supernatant then can be removed by carefully siphoning to a point just above the level of the settled gel. The gel can then be slurried with a minimum volume of homogenization buffer and transferred to a porous support such as a sintered glass funnel. Alternatively, the gel can be collected by centrifugation (e.g., in a Sharples centrifuge) immediately after mixing and packed on a support. After the gel has packed, it is washed with 2 liters of ureacontaining buffer to remove any trapped supernatant, followed by washing with 3 liters of buffer C. The gel is then eluted with 5 liters of buffer C containing 0.5 M NaC1 and concentrated to 800 ml by high-volume ultrafiltration (120 ml/min; Pellicon cassette system, PTGC membrane, Millipore Corp.).
Step 3: Organomercurial Agarose/Melittin-Sepharose Chromatography Steps. The concentrated anion-exchange eluate is made 2 mM in calcium (16 ml of 0.1 M CaC1) and a 200-ml portion of this is applied to a bed of organomercurial agarose (320 ml on a 600-ml sintered glass funnel) which was previously equilibrated in buffer A. The effluent from this step is passed directly through a column of melittin-Sepharose (120 ml in a
16
ISOLATION/CHARACTERIZATION OF Ca-BINDING PROTEINS
[1]
4-cm-diameter column) equilibrated in buffer A. Successive 200-ml additions are made at intervals which allow effluent to pass completely through the melittin-Sepharose. After sample application is complete, the Affi-Gel 501 is washed twice with 200 ml portions of buffer A, again directly onto the column of melittin-Sepharose. The two affinity gels are then separated and each washed with two bed volumes of buffer A containing 0.5 M NaCI. To the washed melittin-Sepharose 75 ml of buffer B are added and the effluent discarded. After 30 min, this gel is eluted with 360 ml of buffer B to yield essentially homogeneous CaM (1.0-1.4 g). The melittin-Sepharose is exhaustively regenerated with low pH urea buffer (see preparation of affinity matrix) followed by equilibration in buffer A. To the washed organomercurial gel (which contains bound S-100 protein) 200 ml of buffer A containing 10 mM dithiothreitol is added and the effluent discarded. The gel is then eluted with four successive 200-ml additions of thiol-containing buffer directly onto the equilibrated melittinSepharose gel as described above. The two affinity gels are then separated and the melittin-Sepharose washed with 240 ml of buffer A containing 0.5 M NaCI. To the washed melittin-Sepharose 75 ml of buffer B is added and the effluent discarded. After 30 min, the gel is eluted with 360 ml of buffer B to yield essentially homogeneous S-100 protein (0.8-1.0 g). Notes. Step 1: Urea used in the homogenization buffer should be deionized just before use to minimize ionic impurities (e.g., cyanates); this can be done by rapidly passing an 8 M urea solution over a mixed bed resin such as Rexyn 1-300 (Fisher). Step 2: It is crucial to wash the packed QAE gel with at least 1.2 bed volumes of urea-containing buffer before proceeding with buffer lacking urea; if this is not done, urea-soluble components in the trapped supernatant precipitate and stop flow through the gel. Step 3: If only CaM is desired, melittin-Sepharose chromatography can be carried out directly, omitting the organomercurial step; the eluate should be passed over a small column (60 ml) of Affi-Gel 501 to remove any contaminating S-100 protein. Although melittin-Sepharose chromatography of supernatant fractions (Method 1) gives excellent results, the use of the urea extraction of brain tissue, followed by a batchwise ion-exchange step prior to melittinSepharose, is preferable for large-scale procedures. Because only rapid, low-speed centrifugation of the homogenate is required, larger amounts of tissue can be processed with relative ease in conventional centrifuges with production of very little insoluble material. Furthermore, homogenization in 6 M urea solubilizes a large amount of brain CaM (40%) which is otherwise difficult to extract from the particulate fraction. The subsequent use of QAE-Sephadex A-25 provides, in addition to an ion-ex-
[1]
17
MELITTIN-SEPHAROSE AFFINITY CHROMATOGRAPHY
! J Q-I
~10.0
;, 3.2
o~
E i
z
iii I0 E:
"
2.4
6.0 B
2.0
¢0 0
÷= ~ Z
1.6
m
,o.8
g -I
3 20
30
40
50
60
70
FRACTION NUMBER Fro. 7. Gel filtration of QAE-Sephadex A-25 eluate. The anion-exchange eluate (30 ml, 45 mg protein/ml), prepared as described for method 2, was chromatographed on a column (94 × 5 cm) of Ultragel AcA 54 (LKB) at a flow rate of 80 ml/hr. Fractions of 20 ml were collected and monitored for protein ( - - A - - ) and A280 ( - - A - - ) . Portions (-10/zg) of the starting material (Q-l) and selected fractions, as indicated by the bent lines, were analyzed by SDS-gel electrophoresis (inset). The two proteins in the major peak of protein are CaM (upper band) and S-100 protein (lower band).
change step, selective binding of smaller proteins and nucleotides; the eluate from this step shows essentially only CaM and S-100 protein with minor amounts of other higher molecular weight proteins (Fig. 7). As noted earlier, when CaM and S-100 protein are both present in the fraction applied to melittin-Sepharose, CaM efficiently displaces S-100 protein, resulting in a small amount of the latter protein in the eluate. Since SI00 protein contains free sulfhydryl groups, while CaM does not, they can be separated quantitatively by using a small column of organomercurial agarose (Fig. 8). The yield of CaM when this procedure is used ranges from 270-350 mg/kg tissue and is the same from bovine, ovine, or porcine brain. One such preparation, from 6 kg tissue is shown in the table. If S100 protein is also desired, the use of the sulfhydryl adsorbent prior to melittin-Sepharose permits preparative isolation of both proteins in com-
18
ISOLATION/CHARACTERIZATION
OF Ca-BINDING PROTEINS
[1]
--94K --67K --43K --30K -21K -- 14K
1
2
3
4
STD
FIG. 8. Separation of CaM and S-100 protein usmg organomercunal agarose chromatography. For this experiment, CaM was prepared by method 2 through elution of the melittinSepharose (prior adsorption of S-100 protein on the sulfhydryl gel was omitted). A portion of the eluate (5 ml, 5 mg of protein) was applied to a column (0.7 x 3.6 cm, 1.5 ml) of organomercurial agarose (Affi-Gel 501, Bio-Rad). This column was washed with 1.2 bed volumes of buffer B, and the effluent was added to the fraction not retained. After washing with three bed volumes of buffer B, the column was eluted with three bed volumes of buffer B containing 10 mM dithiothreitol. Proteins were precipitated and solubilized for SDS-gel electrophoresis. Lane 1, 5/xl of starting material; lane 2, 7/zl of material not retained on organomercurial agarose (CaM); lane 3, 5/~1 of the wash fraction; lane 4, 5/~1 of the eluate (S-100 protein). Coomassie blue-stained material in the region of 60-70 kDa is a gel artifact, since it was found in lanes which contained no protein. Reprinted by permission from Ref. 1.
[2]
CALCIMEDINS
19
TABLE I LARGE-SCALE PURIFICATION OF CALMODUL1N AND S-100 PROTEIN a
Preparation 1 (bovine brain)
Step Homogenate QAE-Sephadex Melittin-Sepharose Eluate 1 (CAM) Melittin-Sepharose Eluate 2 (S-100)
Preparation 2 (ovine brain)
Volume (ml)
Protein (mg)
Yield (mg/kg tissue)
Volume (ml)
Protein (rag)
Yield (mg/kg tissue)
27,600 1,040 540
-2,920 1,920 b
l -320
17,800 800 360
-2,180 1,350 b
--338
--
--
--
1,080
270
360
a The purification procedure was essentially as outlined under method 2 starting with either 6 kg (preparation 1) or 4 kg (preparation 2) of brain tissue. In preparation 1, the large-scale organomercurial agarose step was omitted and the QAE-Sephadex eluate applied directly to the melittin-Sepharose; the eluate from this latter step was depleted of a small amount of S-100 by passage over a column of Affi-Gel 501 (see Fig. 8). Estimates of protein were made using the G-250 dye-binding assay (Bio-Rad) with bovine serum albumin as standard. b Protein for calmodulin determined using purified calmodulin as standard.
parable yield (Table I). Because of the speed, reproducibility, and scaleup potential of this procedure, it should prove useful for investigators involved in physical and structural studies of the two proteins where large quantities of protein are necessary.
[2] C a l c i m e d i n s : P u r i f i c a t i o n a n d P r o d u c t i o n o f A n t i b o d i e s B y J . K . M A T H E W , R . R . SCULLY, V . L . S M I T H , E . BERNICKER, a n d J. R. DEDMAN
Physiological responses in excitable cells are often preceded by an elevation of free calcium. ~This intracellular "calcium signal" is mediated by high-affinity, metal-selective protein receptors. 2 The first such protein mediator identified was the troponin complex of striated muscle) One I H. Rasmussen, in "Calcium and Cell Function" (W. Y. Cheung, ed.), Vol. 4, p. 1. Academic Press, New York, 1983. 2 A. R. Means and J. R. Dedman, Nature (London) 285, 73 (1980). 3 p. B. Moore and J. R. Dedman, J. Biol. Chem. 257, 9663 (1982).
METHODS IN ENZYMOLOGY, VOL. 139
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.