- Email: [email protected]
[9] Purification of bacterial luciferase by affinity methods
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for which genes the mutant phenotypes were not known. For example, transposon mini-Mulux mutants containing insertions in genes regulated by calcium were isolated by replica plating the bank onto nutrient plates with and without added calcium and selecting those strains which only produced light in response to addition of calcium. The regulation of light production in such a strain is shown in Fig. 6. The target gene, of unknown function, was fused to the lux genes which were expressed when calcium induced transcription of the target gene. Genetic study can now be commenced to define the function of this previously unrecognized gene. A similar approach could be used to identify genes regulated by cAMP, nutrient limitation, or a variety of other factors. Furthermore, now that the lux genes have been made mobile by inserting them in a transposon, many kinds of nonluminous bacteria could be tagged with the distinctive Lux phenotype. Strains marked with luminescence could be useful for following the fate of bacteria released into the environment. Acknowledgment The authors acknowledge support of their work at The Agouron Institute by a contract from the Office of Naval Research.
[9] Purification of B a c t e r i a l L u c i f e r a s e b y A f f i n i t y M e t h o d s By THOMAS O. BALDWIN, THOMAS F. HOLZMAN, RITA S . HOLZMAN, and VICKI A. RIDDLE Any purification method requires an accurate and reproducible assay, and the various reported approaches to the purification of bacterial luciferase all benefit from the rapid, reproducible, and precise assays that are available. Bacterial luciferase catalyzes the reaction shown in Fig. 1. The various commonly used assays, which have been summarized in a previous volume of this series, ~differ only in the methods used to supply the substrates FMNH2, aldehyde, and 02. The conditions of each type of assay, as well as the intensity of the light detected, reflect specific aspects of the interactions of the luciferase with its substrates.: Understanding of these interactions is important to an understanding of the rationale of the various methods; we therefore review several important considerations here. J. W. Hastings, T. O. Baldwin, and M. Ziegler-Nicoli, this series, Vol. 57, p. 135. z M. M. Ziegler and T. O. Baldwin, Curr. Top. Bioenerg. 12, 65 (1981).
METHODS IN ENZYMOLOGY, VOL. 133
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
[9]
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PURIFICATION OF BACTERIAL LUCIFERASE
FMNH 2 * E .
" E'FH 2
02
02
•
RCHO " E.FHOOH . ":E'FHOOH
H202
~ " - RCOOH [E.FHOH*]
FMN ÷ H202
~'E'~--FMN
h"d490n m
E'FHOH
+ H20
FIG. 1. Proposed reaction pathway for the bacterial luciferase reaction.
The FMNH~ Injection Assay The most commonly used assay for routine measurements is the FMNH2 injection assay. The reaction is initiated by injection of reduced flavin (generally catalytically reduced by H2 over platinized asbestos) into a vial containing enzyme, aldehyde, and 02. The FMNH2 binds to the luciferase (forming intermediate I) and reacts with 02 to form the 4aperoxydihydroflavin intermediate (intermediate II), 3 which in the presence of bound aldehyde substrate (intermediate IIA), then reacts with the aldehyde to form an excited flavin species and the carboxylic acid product. The excited flavin species emits a photon of blue-green light; ground state FMN is the flavin product of the reaction. Any reduced flavin that does not bind to the enzyme to form intermediate II by reaction with 02 will react nonenzymically with 02 to yield FMN and H202. The substrate is thereby removed from the reaction much more rapidly than the ratelimiting step in the catalytic cycle, so no catalytic turnover is possible in this assay. The activity of the enzyme is measured as the peak intensity of light emission reached following the injection of FMNH2 (Fig. 2). In the absence of further FMNH2 substrate, the emission intensity then decays in a first-order fashion, the rate of decay reflecting the rate-limiting step in the pathway after formation of intermediate IIA. The peak intensity with saturating substrate concentrations is proportional to the amount of active enzyme present in the reaction over at least six orders of magnitude in enzyme concentration, but the decay rate of 3 j. W. Hastings, C. Balny, C. LePeuch, and P. Douzou, Proc. Natl. Acad. Sci. U.S.A. 70, 3468 (1973).
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i
[9]
i
i
60
80
100
80 v lZ IJJ l-Z I-. "r
60
40
o M
,--I
0
0
K
20
I
40
100
4 FMNH2
TIME (sec)
FIG. 2. Time course of the FMNH2 injection luciferase assay. The assay was initiated by injection of 1.0 ml of catalytically reduced flavin mononucleotide into a vial containing luciferase and n-decanal [20 /~l of a 0.01% (v/v) sonicated suspension] in 1.0 ml of airequilibrated buffer (pH 7.0) containing 0.2% BSA (w/v). Light intensity was monitored as a function of time following injection of FMNH2.
the light emission is independent of the concentration of enzyme4; rather, as indicated above, the decay of the luminescence reflects the rate-determining step in the catalytic pathway. While the decay rate is strongly affected by the chain length of the aldehyde substrate, the quantum yield of the reaction (proportional to the area under the emission curve) is independent of the chain length of the aldehyde for chain lengths greater than 6 carbons: the initial maximum light intensity is high if the decay rate is rapid and low if the decay rate is slow) The concentration of aldehyde in the assay mixture is of critical importance, since the enzyme from Vibrio harveyi is subject to aldehyde substrate inhibition) ,6 The peak light emission is not only dependent on the concentration of aldehyde, but on the buffer composition. In buffers containing high concentrations of phosphate (-0.2 M), the luciferase is much less sensitive to aldehyde inhibition than in low phosphate (-0.02 M) buffers or cationic buffers such as Bis-Tris. Addition of bovine serum 4 j. W. Hastings, Q. H. Gibson, J. Friedland, and J. Spudich, In "Bioluminescence in Progress" (F. H. Johnson and Y. Haneda, eds.), pp. 151-186. Princeton University Press, Princeton, N.J., 1966. 5 j. W. Hastings, K. Weber, J. Friedland, A. Eberhard, G. W. Mitchell, and A. Gunsalus, Biochemistry 8, 4681 (1969). 6 T. F. Holzman and T. O. Baldwin, Biochemistry 22, 2838 (1983).
[9]
PURIFICATION OF BACTERIAL LUCIFERASE
101
albumin (BSA) to the assay buffer also protects the enzyme from aldehyde inhibition, possibly due to the aldehyde buffering effect of the protein. The enzyme-FMNH2 complex does not appear to be susceptible to aldehyde inhibition. The affinity purification method that we describe here is based on the cooperative interactions of the enzyme luciferase with the inhibitor 2,2diphenylpropylamine (D~bPA) and phosphate. 7,8 The inhibitor is competitive with the substrate aldehyde, but binds more tightly to the enzymeFMNH~ complex. Likewise, the inhibitor binds more tightly to the enzyme-phosphate complex than to the "free" enzyme. The impure enzyme in a high phosphate buffer can be bound to the inhibitor attached to an insoluble support material (Sepharose); impurities can be removed by washing with the high phosphate buffer. Subsequently switching to a buffer without phosphate reduces the affinity between the enzyme and the inhibitor, releasing the enzyme from the support. This selective ternary complex formation allows for a very high level of purification. 8 We give the detailed procedures below.
Reagents and Materials Required for D~bPA-Sepharose Synthesis Sepharose (either 4B or 6B) 1,4-Butanediol diglycidyl ether (Aldrich) 2,2-Diphenylpropylamine (Aldrich; 10 mg/ml settled volume of Sepharose) 0.6 M NaOH (1 ml/ml settled volume of Sepharose) NaBH4 Dioxane/0.2 M sodium carbonate, pH 10.5 (1 : 1, v/v) Dioxane/water (1 : 1) Dioxane/0.20 M phosphate, pH 7.0 (1 : 1) 95% ethanol Sintered glass funnel Rotary evaporator with regulated water bath (Buchi model Rotavapot R, or equivalent) Round-bottomed flask (at least four times the settled volume of Sepharose) Synthesis of Dq~PA-Sepharose To 100 ml (settled volume) of Sepharose 6B in a 500-ml round-bottomed flask was added 100 ml of 1,4-butanediol diglycidyl ether, 100 ml 0.6 M NaOH, and 200 mg sodium borohydride. The flask was attached to a rotary evaporator and the slurry mixed by slow rotation overnight at 7 T. F. Holzman and T. O. Baldwin, 8 T. F. Holzman and T. O. Baldwin,
Biochemistry 20, 5524 Biochemistry 24, 6194
(1981). (1982).
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room temperature. The resulting epoxy-activated Sepharose was collected on a sintered glass funnel, washed to neutrality with water, and suction dried. The ligand 2,2-diphenylpropylamine was dissolved at 10 mg/ml in dioxane/0.2 M sodium carbonate, pH 10.5 (1 : 1, v/v). The coupling of the ligand to the epoxy-activated Sepharose was initiated by mixing 100 ml of the 2,2-diphenylpropylamine solution with 100 ml (settled volume) of the epoxy-activated Sepharose in a 500-ml round-bottomed flask. The flask was again attached to the rotary evaporator and the slurry mixed by slow rotation at 60 ° for about 24 hr. As the reaction proceeded, the pH of the solution dropped. To maintain a reasonable rate of reaction, the pH was checked and adjusted to 10.5 with 0.1 M NaOH every 3 hr for the first 12 hr, and again after 18 hr. The pH did not change between 18 and 24 hr, indicating that the reaction was complete. After the 24-hr coupling reaction, the D~bPA-Sepharose (-100 ml) was collected on a sintered glass funnel and washed successively with 600 ml of 1 : 1 dioxane/water, 600 ml of 1 : 1 dioxane/0.20 M phosphate, pH 7.0, and 1200 ml of 95% ethanol. The D~bPA-Sepharose was then either used directly or stored in 50% ethanol/water at 4 ° (Fig. 3). Preparation of Luciferase for Binding to the DthPA-Sepharose Lysis of bacterial cells was accomplished as described in detail previously. 1 There are several key points to note regarding cell lysis. First, freezing cells in liquid nitrogen or in a low temperature ( - 7 0 ° or lower) freezer results in poor lysis, probably because ice crystal formation is reduced by rapid freezing. Rather, slow freezing at - 2 0 ° and storage in a non-frost-free freezer results in the best lysis. Storage and thawing are best accomplished in thin pads of cell paste in freezer bags. On rare occasions in which we have thawed and refrozen cells at - 2 0 °, we have obtained the most rapid and complete lysis. Cell Lysis The general procedure to lyse the cells by osmotic shock is presented below. The procedure is carried out at 4° . 1. Remove the bag of cells from the freezer and allow to thaw for several hours at room temperature or overnight at 4 °. Add a small volume of 3% NaCI to the thawed cell paste and stir well, such that the cell paste becomes sufficiently fluid that it may be easily poured. 2. Slowly pour the cell paste into rapidly stirred deionized H20 that is about 4 °. The volume of water should be about 5 ml/g of original cell
[9]
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P U R I F I C A T I O N OF B A C T E R I A L L U C I F E R A S E
1,4-Diglycidyl
Ether
/o\
/0\
OH + H 2 C - C H - C H 2 - O - ( C H 2 ) 4 - O - C H 2 - C H - C H
2
A c t i v a t i o n and C r o s s l i n k i n g
Sepharose Gel B e a d
Suction Dried Sepharose ( l g ) is washed on • glass filter with water and mixed with lml of 1,4-diglycidyl ether, lml of 0.6 ~ NaOH, and 2rag of NaBH4. The suspension was mixed by rotation in a round-bottomed flask for ~ 8 hours at room t e m p e r a t u r e . The r e a c t i o n was stopped by washing the material with large volumes of water.
H
/O\
O-CH2-CH-CH2-O-(CH2)
4-O-cH2-cH-cH
•
I
z+ HzN-CH2-?-CH ~ O
L i g a n d Couplin~l
2,2 - D i p h e n y l p r o p y l a m i n e
For e a c h gram of e p o x y - a c t i v a t e d Sepharose, lml of a 10mglml solution of 2,2-dlphenylpropylamlne in dioxane/0.2M sodium c a r b o n a t e , pH 10.5 ( f : f , v : v ) , was added and the slurry mixed by rotation In a round-bottomed flask at 60" for 24 hours.
OH e H I I O-CH2-CH-CH2-O(CH2) 4-O-CH~-CH-CH~-NH-CH~-C-CH:3 0
DOPA-Sepharose FIc. 3. Flowchart for the synthesis of D&PA-Sepharose. The details of the procedure are given in the text.
paste, and it is critical that the water be stirred as rapidly as can be accomplished with a magnetic stirrer, so that the cells can be dispersed quickly. Failure to stir the lysis mixture will result in poor lysis, apparently due to equilibration of the internal and external salt concentration without cell lysis. Addition of the cell paste to the stirred water should be done in small aliquots such that the complete addition of 500 g of cell paste to 2.5 liters would be complete in about 20 rain. The pH of the lysate should be checked regularly during the addition of cell paste; the pH normally drops and should be maintained at about pH 7 by addition in small increments of Tris base to as much as 1 g/liter. 3. Lysis is usually complete within 15 min of the final addition of cells to the water. To check for complete lysis, it is important to remove the cell debris from the solution. Luciferase in unlysed cells will react and emit light in the flavin injection assay. Lysis is complete if the luciferase activity in 10 ~1 of supernatant following centrifugation for 1 min in an
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Eppendorf microcentrifuge is within 10% of the activity of 10/zl of the uncentrifuged lysate. 4. If complete lysis does not occur, one must resort to sonication. Since the enzyme from all species studied is sensitive to oxidation, DTE should be added to about 0.5 m M prior to sonication. The only times that we have had serious trouble with cell lysis were when we tried to lyse fresh cells directly from the fermenter or cells that had only been frozen overnight. It appears that after a week or more in the freezer, the cells become more fragile and subject to lysis by osmotic shock. Ammonium Sulfate Fractionation If the standard purification method is to be employed,l,9 DEAE-cellulose batch extraction is performed at this stage. The affinity purification methods described here, using either the column approach or the batch approach, require NH4SO4 fractionation of the lysate at this stage. The initial volume of the uncentrifuged lysate is measured and recorded. Solid ammonium sulfate is added to the lysate (at 0-4 °) slowly, such that addition of 100 g of ammonium sulfate requires about 5 rain. For the V. harveyi lysates, the fraction precipitating between 40 and 80% saturation of ammonium sulfate is collected; for the Vibriofischeri and Photobacterium phosphoreum, the fractions precipitating between 35 and 80% ammonium sulfate are collected. Ammonium sulfate (242 g/1000 ml of initial volume for V. harveyi and 209 g/1000 ml of initial volume for V. fischeri and P. phosphoreum) is added to the uncentrifuged lysates and stirred slowly with a magnetic stir bar for about 15 rain at 0-4 °. The suspension is then clarified by centrifugation at 16,000 g (9500 rpm in a Beckman JA-10 rotor) for 30 min at 4 °. The supernatants are collected and solid ammonium sulfate (318 g/1000 ml initial volume for V. harveyi and 351 g/1000 ml initial volume for V. fischeri and P. phosphoreum) is added as before. After about 15 min of slow stirring at 0-4 °, the precipitated protein is collected by centrifugation as before. The precipitated protein is removed from the centrifuge bottles with a stainless-steel iced tea spoon and delivered to dialysis tubing through a small glass powder funnel. The remaining precipitated protein is washed out of the bottles and delivered to the dialysis tubing with a minimal amount of 0.1 M phosphate, pH 7.0, such that the protein concentration following dialysis will be 50 mg/ml or greater. If the sample is to be stored for some time prior to purification of the luciferase, it is best to store it in the presence of - 0 . 5 mM DTE as a 9 A. Gunsalus-Migucl, E. A. Mcighcn, M. Z. Nicoli, K. H. Nealson, and J. W. Hastings, J. Biol. Chem. 247, 398 (1972).
[9]
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PURIFICATION OF BACTERIAL LUCIFERASE APPLICATION AND ELUTION BUFFERS FOR AFFINITY CHROMATOGRAPHYOF LUCIFERASES FROM V. harveyi, V. fischeri, AND P. phosphoreum
Bacterial strain
V. harveyi
V. fischeri P. phosphoreum
Application buffer
Elution buffer
0.10 M phosphate, pH 8.5, 0.50 M NaCl, 0.50 M KCI, 0.5 mM DTE 0. l0 M phosphate, pH 7.0, 0.5 m M DTE 0.35 M phosphate, pH 7.0, 0.5 m M DTE
25 mM ethanolamine, 5.0 mM Tris, pH 9.1, 0.5 mM DTE 0.30 M Tris, pH 8.5, 0.5 mM DTE 0.10 M Tris, pH 8.1, 0.5 mM DTE
frozen ammonium sulfate paste (for V. harveyi and V. fischeri). The luciferases from V. harveyi and V. fischeri are stable for over a year as ammonium sulfate pastes at - 2 0 °. The luciferase from P. phosphoreum is unstable to freezing whether in ammonium sulfate or in buffer, and is best stored for long times in 0.2 M phosphate, pH 7.0, 0.5 mM DTE, 40% glycerol at - 2 0 °. Prior to application to the affinity matrix, the ammonium sulfate is removed from the protein by dialysis against sample application buffer (see the table). The sample is dialyzed against three changes of a 10-fold volume excess of the application buffer and the buffer from the last dialysis is used to equilibrate the affinity matrix prior to sample application. This latter point is of critical importance. We have found that equilibration of the affinity matrix with the buffer with which the sample was last equilibrated results in significantly higher yields and higher binding capacity than are observed when the affinity matrix is equilibrated with "fresh" buffer. Use of DcbPA-Sepharose in a Column Mode The approach to affinity purification of bacterial luciferases comprises a simple " o n - o f f " strategy that is based on the observation that the binding of lucifcrases to 2,2-diphenylpropylamine is enhanced by binding of multivalent anions, especially phosphate, sulfate, and arsenate. 7,s The procedure for all bacterial luciferases is essentially the same, with variation only in the composition of the application and elution buffers. The sample in application buffer (which contains phosphate) is loaded onto a column of the affinity matrix until the column is nearly saturated with the luciferase. The addition of lucifcrase is then stopped and the column washed with application buffer (the same buffer with which the column was equilibrated, i.e., the buffer used in the final dialysis equilibration) until the absorbance at 280 nm and luciferase activity in the effluent have
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dropped to near-baseline values. The column is then washed with elution buffer to remove the luciferase. The luciferases are significantly less stable in the elution buffers than in the application buffers; to circumvent the problem of the luciferase denaturing in the test tubes in the fraction collector used to collect the effluent from the column, a small amount (about 0.1 ml, depending on fraction size) of 1 M phosphate, pH 7.0, is added to each tube in the fraction collector so that the pH of the effluent will drop quickly to near neutrality and the enzyme will be afforded anion stabilization. Affinity Purification of V. harveyi Luciferase In a typical purification using a column with a 30 ml bed volume, the column is preequilibrated with about 100 ml of sample application buffer (used in the final dialysis of the sample) at a flowrate of 40 ml/hr maintained with a peristaltic pump. A typical sample would have an absorbance of 40-50 at 280 nm and 25,000 to 50,000 LU/ml luciferase activity (assayed with n-decanal; 1 L U (light unit) = 9.8 × 109 quanta/sec). The centrifuged (to remove material precipitating during dialysis) sample is applied to the column through the peristaltic pump at a flow rate of about 40 ml/hr. Fractions are collected from the effluent and assayed for protein (absorbance at 280 nm) and for luciferase. When the luciferase activity in the effluent reaches about 5-10% of the activity in the sample, application is terminated. A typical column of 30 ml bed volume can accommodate about 4.0 × 10 6 total light units, and loading of the sample usually requires between 1.5 and 2 hr. After sample loading, the column is washed with the same application buffer with which it had been equilibrated, also at about 40 ml/hr, until the absorbance and luciferase activity have returned to near-baseline values, usually about 4 hr. Luciferase elution is then effected by application of the elution buffer (see the table). The luciferase is eluted at a slower flow rate, about 5-10 ml/hr, to maintain a sharp elution profile. The luciferase elutes from the column in a sharp peak centered about 2-3 column volumes from the initiation of application of the elution buffer. Elution of the luciferase requires about 10-15 hr. While the elution can be carried out much more rapidly, both yields and purity suffer. By the procedure described here, we routinely achieve, by conservative pooling of fractions from the column, luciferase from V. harveyi of about 95% purity with overall yields of 50-75%. Affinity Purification of V. fischeri Luciferase The affinity method works well for both large scale and small scale purifications. To demonstrate the point, we will describe the purification
[9]
P U R I F I C A T I O N OF B A C T E R I A L L U C I F E R A S E
107
of luciferase from 5 g of cell paste on a 1 ml column of D~bPA-Sepharose 6B. The column was preequilibrated as fast as allowed by the gel bed, about 50 ml/hr. The dialyzed sample from the 35-80% ammonium sulfate fractionation (13 ml with absorbance at 280 nm of about 40 and luciferase activity of 25,000 LU/ml assayed with n-dodecanal) was applied to the column in 1-ml aliquots and allowed to enter the bed as fast as possible. The luciferase activity and 280 nm absorbance of the effluent from the column were determined for each addition. The luciferase activity per unit volume in the effluent after the final addition was about 5% that of the original sample. The gel bed was then washed with seven 1.0-ml aliquots of application buffer, after which the absorbance at 280 nm and luciferase activity in the effluent were both near baseline. The luciferase was e|uted from the column by successive additions of 1.0-ml aliquots of elution buffer (see the table). Over half of the luciferase activity eluted in the second fraction, which by electrophoresis was estimated to be greater than 95% pure. By this small scale approach, the entire affinity purification requires less than 2 hr. The luciferase from P. phosphoreurn is readily purified by the same approach. The comparative instability of the luciferase from P. phosphoreum requires that care be taken in addition of fresh DTE to all buffers and that the eluent from the column be mixed immediately with ! M phosphate, pH 7.0. Otherwise, low yields are routinely observed. Use of D~bPA-Sepharose in a Batch Mode For very large scale rapid purifications of bacterial luciferase with yields that suffer only slightly compared with the ion exchange or affinity column methods, a batch affinity method may be employed. The sample preparation for the batch and column methods is identical. The affinity matrix is equilibrated with the application buffer (again, that used for the final dialysis) on a coarse sintered glass funnel. The buffer is added to the gel in aliquots and then suction dried by aspirator vacuum. The suction is turned off after each drying step, more buffer added and allowed to drip through, and then dried again by application of the vacuum. Equilibration of 500 g (suction-dried weight) of the gel requires about 1 liter of buffer. The suction dried equilibrated D~bPA-Sepharose on the sintered glass funnel is then titrated with the dialyzed sample after ammonium sulfate fractionation of the lysate obtained from about 500 g of V. harveyi cells. The sample volume from 500 g of cells is usually about 500-600 ml; aliquots of 100 ml are added to the gel and allowed to drip through. The gel is then dried after each addition by a short application of the aspirator vacuum. The luciferase activity is determined from the eluate after each
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addition, and the gel is considered to be saturated when the activity per unit volume in the eluate reaches about 5% that of the original sample. The capacity of the gel for pure luciferase from V. harveyi is about 8 mg/ ml gel; the capacity is about 2 mg/ml gel for luciferase that has been partially purified by ammonium sulfate fractionation. The luciferase content of 500 g of V. harveyi cells is about 1 g. After sample application is complete, the gel is washed on the sintered glass funnel with four to five 100-ml aliquots of the application buffer. The absorbance at 280 nm and the luciferase activity in the eluate are determined after each addition. The wash steps are performed as before; the buffer is applied without vacuum to the gel, allowed to drip through, and the gel is dried by a short application of the aspirator vacuum. When the absorbance at 280 nm and the luciferase activity in the eluate are low (A280 < 0.2), the luciferase is eluted by successive additions of the elution buffer (see the table). Elution usually requires seven 100-ml aliquots of buffer. Before each addition, 10 ml of 1.0 M phosphate, pH 7.0, is added to the vacuum flask so that the pH of the eluate will drop to near neutrality and the luciferase will be stabilized by the phosphate as it elutes from the gel. The elution buffer is added to the gel and allowed to drip through before application of the aspirator. Following drying of the gel after each 100-ml aliquot of elution buffer, the funnel is transferred to a fresh vacuum flask containing 10 ml of 1.0 M phosphate, pH 7.0. Each 110 ml fraction is assayed for luciferase activity and the absorbance at 280 nm is determined. Aliquots with the highest specific activity are pooled, concentrated by precipitation with ammonium sulfate at 80% saturation, and either dialyzed for experimental use or frozen as an ammonium sulfate paste. The batch method routinely yields about 60% of the luciferase in the dialyzed sample at a purity level of better than 90%. The overall batch method requires only about 3 hr, and from 500 g of V. harveyi cell paste, a yield of about 600-700 mg of luciferase is obtained. Acknowledgments This research was supported by grants from the National Science Foundation (PCM 8208589), the National Institutes of Health (AG 03697), the Robert A. Welch Foundation (A-865), the Upjohn Company, and a Hatch Grant (RI 6545).
BIOLUMINESCENCE
[9]
for which genes the mutant phenotypes were not known. For example, transposon mini-Mulux mutants containing insertions in genes regulated by calcium were isolated by replica plating the bank onto nutrient plates with and without added calcium and selecting those strains which only produced light in response to addition of calcium. The regulation of light production in such a strain is shown in Fig. 6. The target gene, of unknown function, was fused to the lux genes which were expressed when calcium induced transcription of the target gene. Genetic study can now be commenced to define the function of this previously unrecognized gene. A similar approach could be used to identify genes regulated by cAMP, nutrient limitation, or a variety of other factors. Furthermore, now that the lux genes have been made mobile by inserting them in a transposon, many kinds of nonluminous bacteria could be tagged with the distinctive Lux phenotype. Strains marked with luminescence could be useful for following the fate of bacteria released into the environment. Acknowledgment The authors acknowledge support of their work at The Agouron Institute by a contract from the Office of Naval Research.
[9] Purification of B a c t e r i a l L u c i f e r a s e b y A f f i n i t y M e t h o d s By THOMAS O. BALDWIN, THOMAS F. HOLZMAN, RITA S . HOLZMAN, and VICKI A. RIDDLE Any purification method requires an accurate and reproducible assay, and the various reported approaches to the purification of bacterial luciferase all benefit from the rapid, reproducible, and precise assays that are available. Bacterial luciferase catalyzes the reaction shown in Fig. 1. The various commonly used assays, which have been summarized in a previous volume of this series, ~differ only in the methods used to supply the substrates FMNH2, aldehyde, and 02. The conditions of each type of assay, as well as the intensity of the light detected, reflect specific aspects of the interactions of the luciferase with its substrates.: Understanding of these interactions is important to an understanding of the rationale of the various methods; we therefore review several important considerations here. J. W. Hastings, T. O. Baldwin, and M. Ziegler-Nicoli, this series, Vol. 57, p. 135. z M. M. Ziegler and T. O. Baldwin, Curr. Top. Bioenerg. 12, 65 (1981).
METHODS IN ENZYMOLOGY, VOL. 133
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
[9]
99
PURIFICATION OF BACTERIAL LUCIFERASE
FMNH 2 * E .
" E'FH 2
02
02
•
RCHO " E.FHOOH . ":E'FHOOH
H202
~ " - RCOOH [E.FHOH*]
FMN ÷ H202
~'E'~--FMN
h"d490n m
E'FHOH
+ H20
FIG. 1. Proposed reaction pathway for the bacterial luciferase reaction.
The FMNH~ Injection Assay The most commonly used assay for routine measurements is the FMNH2 injection assay. The reaction is initiated by injection of reduced flavin (generally catalytically reduced by H2 over platinized asbestos) into a vial containing enzyme, aldehyde, and 02. The FMNH2 binds to the luciferase (forming intermediate I) and reacts with 02 to form the 4aperoxydihydroflavin intermediate (intermediate II), 3 which in the presence of bound aldehyde substrate (intermediate IIA), then reacts with the aldehyde to form an excited flavin species and the carboxylic acid product. The excited flavin species emits a photon of blue-green light; ground state FMN is the flavin product of the reaction. Any reduced flavin that does not bind to the enzyme to form intermediate II by reaction with 02 will react nonenzymically with 02 to yield FMN and H202. The substrate is thereby removed from the reaction much more rapidly than the ratelimiting step in the catalytic cycle, so no catalytic turnover is possible in this assay. The activity of the enzyme is measured as the peak intensity of light emission reached following the injection of FMNH2 (Fig. 2). In the absence of further FMNH2 substrate, the emission intensity then decays in a first-order fashion, the rate of decay reflecting the rate-limiting step in the pathway after formation of intermediate IIA. The peak intensity with saturating substrate concentrations is proportional to the amount of active enzyme present in the reaction over at least six orders of magnitude in enzyme concentration, but the decay rate of 3 j. W. Hastings, C. Balny, C. LePeuch, and P. Douzou, Proc. Natl. Acad. Sci. U.S.A. 70, 3468 (1973).
100
BIOLUMINESCENCE i
i
[9]
i
i
60
80
100
80 v lZ IJJ l-Z I-. "r
60
40
o M
,--I
0
0
K
20
I
40
100
4 FMNH2
TIME (sec)
FIG. 2. Time course of the FMNH2 injection luciferase assay. The assay was initiated by injection of 1.0 ml of catalytically reduced flavin mononucleotide into a vial containing luciferase and n-decanal [20 /~l of a 0.01% (v/v) sonicated suspension] in 1.0 ml of airequilibrated buffer (pH 7.0) containing 0.2% BSA (w/v). Light intensity was monitored as a function of time following injection of FMNH2.
the light emission is independent of the concentration of enzyme4; rather, as indicated above, the decay of the luminescence reflects the rate-determining step in the catalytic pathway. While the decay rate is strongly affected by the chain length of the aldehyde substrate, the quantum yield of the reaction (proportional to the area under the emission curve) is independent of the chain length of the aldehyde for chain lengths greater than 6 carbons: the initial maximum light intensity is high if the decay rate is rapid and low if the decay rate is slow) The concentration of aldehyde in the assay mixture is of critical importance, since the enzyme from Vibrio harveyi is subject to aldehyde substrate inhibition) ,6 The peak light emission is not only dependent on the concentration of aldehyde, but on the buffer composition. In buffers containing high concentrations of phosphate (-0.2 M), the luciferase is much less sensitive to aldehyde inhibition than in low phosphate (-0.02 M) buffers or cationic buffers such as Bis-Tris. Addition of bovine serum 4 j. W. Hastings, Q. H. Gibson, J. Friedland, and J. Spudich, In "Bioluminescence in Progress" (F. H. Johnson and Y. Haneda, eds.), pp. 151-186. Princeton University Press, Princeton, N.J., 1966. 5 j. W. Hastings, K. Weber, J. Friedland, A. Eberhard, G. W. Mitchell, and A. Gunsalus, Biochemistry 8, 4681 (1969). 6 T. F. Holzman and T. O. Baldwin, Biochemistry 22, 2838 (1983).
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PURIFICATION OF BACTERIAL LUCIFERASE
101
albumin (BSA) to the assay buffer also protects the enzyme from aldehyde inhibition, possibly due to the aldehyde buffering effect of the protein. The enzyme-FMNH2 complex does not appear to be susceptible to aldehyde inhibition. The affinity purification method that we describe here is based on the cooperative interactions of the enzyme luciferase with the inhibitor 2,2diphenylpropylamine (D~bPA) and phosphate. 7,8 The inhibitor is competitive with the substrate aldehyde, but binds more tightly to the enzymeFMNH~ complex. Likewise, the inhibitor binds more tightly to the enzyme-phosphate complex than to the "free" enzyme. The impure enzyme in a high phosphate buffer can be bound to the inhibitor attached to an insoluble support material (Sepharose); impurities can be removed by washing with the high phosphate buffer. Subsequently switching to a buffer without phosphate reduces the affinity between the enzyme and the inhibitor, releasing the enzyme from the support. This selective ternary complex formation allows for a very high level of purification. 8 We give the detailed procedures below.
Reagents and Materials Required for D~bPA-Sepharose Synthesis Sepharose (either 4B or 6B) 1,4-Butanediol diglycidyl ether (Aldrich) 2,2-Diphenylpropylamine (Aldrich; 10 mg/ml settled volume of Sepharose) 0.6 M NaOH (1 ml/ml settled volume of Sepharose) NaBH4 Dioxane/0.2 M sodium carbonate, pH 10.5 (1 : 1, v/v) Dioxane/water (1 : 1) Dioxane/0.20 M phosphate, pH 7.0 (1 : 1) 95% ethanol Sintered glass funnel Rotary evaporator with regulated water bath (Buchi model Rotavapot R, or equivalent) Round-bottomed flask (at least four times the settled volume of Sepharose) Synthesis of Dq~PA-Sepharose To 100 ml (settled volume) of Sepharose 6B in a 500-ml round-bottomed flask was added 100 ml of 1,4-butanediol diglycidyl ether, 100 ml 0.6 M NaOH, and 200 mg sodium borohydride. The flask was attached to a rotary evaporator and the slurry mixed by slow rotation overnight at 7 T. F. Holzman and T. O. Baldwin, 8 T. F. Holzman and T. O. Baldwin,
Biochemistry 20, 5524 Biochemistry 24, 6194
(1981). (1982).
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room temperature. The resulting epoxy-activated Sepharose was collected on a sintered glass funnel, washed to neutrality with water, and suction dried. The ligand 2,2-diphenylpropylamine was dissolved at 10 mg/ml in dioxane/0.2 M sodium carbonate, pH 10.5 (1 : 1, v/v). The coupling of the ligand to the epoxy-activated Sepharose was initiated by mixing 100 ml of the 2,2-diphenylpropylamine solution with 100 ml (settled volume) of the epoxy-activated Sepharose in a 500-ml round-bottomed flask. The flask was again attached to the rotary evaporator and the slurry mixed by slow rotation at 60 ° for about 24 hr. As the reaction proceeded, the pH of the solution dropped. To maintain a reasonable rate of reaction, the pH was checked and adjusted to 10.5 with 0.1 M NaOH every 3 hr for the first 12 hr, and again after 18 hr. The pH did not change between 18 and 24 hr, indicating that the reaction was complete. After the 24-hr coupling reaction, the D~bPA-Sepharose (-100 ml) was collected on a sintered glass funnel and washed successively with 600 ml of 1 : 1 dioxane/water, 600 ml of 1 : 1 dioxane/0.20 M phosphate, pH 7.0, and 1200 ml of 95% ethanol. The D~bPA-Sepharose was then either used directly or stored in 50% ethanol/water at 4 ° (Fig. 3). Preparation of Luciferase for Binding to the DthPA-Sepharose Lysis of bacterial cells was accomplished as described in detail previously. 1 There are several key points to note regarding cell lysis. First, freezing cells in liquid nitrogen or in a low temperature ( - 7 0 ° or lower) freezer results in poor lysis, probably because ice crystal formation is reduced by rapid freezing. Rather, slow freezing at - 2 0 ° and storage in a non-frost-free freezer results in the best lysis. Storage and thawing are best accomplished in thin pads of cell paste in freezer bags. On rare occasions in which we have thawed and refrozen cells at - 2 0 °, we have obtained the most rapid and complete lysis. Cell Lysis The general procedure to lyse the cells by osmotic shock is presented below. The procedure is carried out at 4° . 1. Remove the bag of cells from the freezer and allow to thaw for several hours at room temperature or overnight at 4 °. Add a small volume of 3% NaCI to the thawed cell paste and stir well, such that the cell paste becomes sufficiently fluid that it may be easily poured. 2. Slowly pour the cell paste into rapidly stirred deionized H20 that is about 4 °. The volume of water should be about 5 ml/g of original cell
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103
P U R I F I C A T I O N OF B A C T E R I A L L U C I F E R A S E
1,4-Diglycidyl
Ether
/o\
/0\
OH + H 2 C - C H - C H 2 - O - ( C H 2 ) 4 - O - C H 2 - C H - C H
2
A c t i v a t i o n and C r o s s l i n k i n g
Sepharose Gel B e a d
Suction Dried Sepharose ( l g ) is washed on • glass filter with water and mixed with lml of 1,4-diglycidyl ether, lml of 0.6 ~ NaOH, and 2rag of NaBH4. The suspension was mixed by rotation in a round-bottomed flask for ~ 8 hours at room t e m p e r a t u r e . The r e a c t i o n was stopped by washing the material with large volumes of water.
H
/O\
O-CH2-CH-CH2-O-(CH2)
4-O-cH2-cH-cH
•
I
z+ HzN-CH2-?-CH ~ O
L i g a n d Couplin~l
2,2 - D i p h e n y l p r o p y l a m i n e
For e a c h gram of e p o x y - a c t i v a t e d Sepharose, lml of a 10mglml solution of 2,2-dlphenylpropylamlne in dioxane/0.2M sodium c a r b o n a t e , pH 10.5 ( f : f , v : v ) , was added and the slurry mixed by rotation In a round-bottomed flask at 60" for 24 hours.
OH e H I I O-CH2-CH-CH2-O(CH2) 4-O-CH~-CH-CH~-NH-CH~-C-CH:3 0
DOPA-Sepharose FIc. 3. Flowchart for the synthesis of D&PA-Sepharose. The details of the procedure are given in the text.
paste, and it is critical that the water be stirred as rapidly as can be accomplished with a magnetic stirrer, so that the cells can be dispersed quickly. Failure to stir the lysis mixture will result in poor lysis, apparently due to equilibration of the internal and external salt concentration without cell lysis. Addition of the cell paste to the stirred water should be done in small aliquots such that the complete addition of 500 g of cell paste to 2.5 liters would be complete in about 20 rain. The pH of the lysate should be checked regularly during the addition of cell paste; the pH normally drops and should be maintained at about pH 7 by addition in small increments of Tris base to as much as 1 g/liter. 3. Lysis is usually complete within 15 min of the final addition of cells to the water. To check for complete lysis, it is important to remove the cell debris from the solution. Luciferase in unlysed cells will react and emit light in the flavin injection assay. Lysis is complete if the luciferase activity in 10 ~1 of supernatant following centrifugation for 1 min in an
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Eppendorf microcentrifuge is within 10% of the activity of 10/zl of the uncentrifuged lysate. 4. If complete lysis does not occur, one must resort to sonication. Since the enzyme from all species studied is sensitive to oxidation, DTE should be added to about 0.5 m M prior to sonication. The only times that we have had serious trouble with cell lysis were when we tried to lyse fresh cells directly from the fermenter or cells that had only been frozen overnight. It appears that after a week or more in the freezer, the cells become more fragile and subject to lysis by osmotic shock. Ammonium Sulfate Fractionation If the standard purification method is to be employed,l,9 DEAE-cellulose batch extraction is performed at this stage. The affinity purification methods described here, using either the column approach or the batch approach, require NH4SO4 fractionation of the lysate at this stage. The initial volume of the uncentrifuged lysate is measured and recorded. Solid ammonium sulfate is added to the lysate (at 0-4 °) slowly, such that addition of 100 g of ammonium sulfate requires about 5 rain. For the V. harveyi lysates, the fraction precipitating between 40 and 80% saturation of ammonium sulfate is collected; for the Vibriofischeri and Photobacterium phosphoreum, the fractions precipitating between 35 and 80% ammonium sulfate are collected. Ammonium sulfate (242 g/1000 ml of initial volume for V. harveyi and 209 g/1000 ml of initial volume for V. fischeri and P. phosphoreum) is added to the uncentrifuged lysates and stirred slowly with a magnetic stir bar for about 15 rain at 0-4 °. The suspension is then clarified by centrifugation at 16,000 g (9500 rpm in a Beckman JA-10 rotor) for 30 min at 4 °. The supernatants are collected and solid ammonium sulfate (318 g/1000 ml initial volume for V. harveyi and 351 g/1000 ml initial volume for V. fischeri and P. phosphoreum) is added as before. After about 15 min of slow stirring at 0-4 °, the precipitated protein is collected by centrifugation as before. The precipitated protein is removed from the centrifuge bottles with a stainless-steel iced tea spoon and delivered to dialysis tubing through a small glass powder funnel. The remaining precipitated protein is washed out of the bottles and delivered to the dialysis tubing with a minimal amount of 0.1 M phosphate, pH 7.0, such that the protein concentration following dialysis will be 50 mg/ml or greater. If the sample is to be stored for some time prior to purification of the luciferase, it is best to store it in the presence of - 0 . 5 mM DTE as a 9 A. Gunsalus-Migucl, E. A. Mcighcn, M. Z. Nicoli, K. H. Nealson, and J. W. Hastings, J. Biol. Chem. 247, 398 (1972).
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PURIFICATION OF BACTERIAL LUCIFERASE APPLICATION AND ELUTION BUFFERS FOR AFFINITY CHROMATOGRAPHYOF LUCIFERASES FROM V. harveyi, V. fischeri, AND P. phosphoreum
Bacterial strain
V. harveyi
V. fischeri P. phosphoreum
Application buffer
Elution buffer
0.10 M phosphate, pH 8.5, 0.50 M NaCl, 0.50 M KCI, 0.5 mM DTE 0. l0 M phosphate, pH 7.0, 0.5 m M DTE 0.35 M phosphate, pH 7.0, 0.5 m M DTE
25 mM ethanolamine, 5.0 mM Tris, pH 9.1, 0.5 mM DTE 0.30 M Tris, pH 8.5, 0.5 mM DTE 0.10 M Tris, pH 8.1, 0.5 mM DTE
frozen ammonium sulfate paste (for V. harveyi and V. fischeri). The luciferases from V. harveyi and V. fischeri are stable for over a year as ammonium sulfate pastes at - 2 0 °. The luciferase from P. phosphoreum is unstable to freezing whether in ammonium sulfate or in buffer, and is best stored for long times in 0.2 M phosphate, pH 7.0, 0.5 mM DTE, 40% glycerol at - 2 0 °. Prior to application to the affinity matrix, the ammonium sulfate is removed from the protein by dialysis against sample application buffer (see the table). The sample is dialyzed against three changes of a 10-fold volume excess of the application buffer and the buffer from the last dialysis is used to equilibrate the affinity matrix prior to sample application. This latter point is of critical importance. We have found that equilibration of the affinity matrix with the buffer with which the sample was last equilibrated results in significantly higher yields and higher binding capacity than are observed when the affinity matrix is equilibrated with "fresh" buffer. Use of DcbPA-Sepharose in a Column Mode The approach to affinity purification of bacterial luciferases comprises a simple " o n - o f f " strategy that is based on the observation that the binding of lucifcrases to 2,2-diphenylpropylamine is enhanced by binding of multivalent anions, especially phosphate, sulfate, and arsenate. 7,s The procedure for all bacterial luciferases is essentially the same, with variation only in the composition of the application and elution buffers. The sample in application buffer (which contains phosphate) is loaded onto a column of the affinity matrix until the column is nearly saturated with the luciferase. The addition of lucifcrase is then stopped and the column washed with application buffer (the same buffer with which the column was equilibrated, i.e., the buffer used in the final dialysis equilibration) until the absorbance at 280 nm and luciferase activity in the effluent have
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dropped to near-baseline values. The column is then washed with elution buffer to remove the luciferase. The luciferases are significantly less stable in the elution buffers than in the application buffers; to circumvent the problem of the luciferase denaturing in the test tubes in the fraction collector used to collect the effluent from the column, a small amount (about 0.1 ml, depending on fraction size) of 1 M phosphate, pH 7.0, is added to each tube in the fraction collector so that the pH of the effluent will drop quickly to near neutrality and the enzyme will be afforded anion stabilization. Affinity Purification of V. harveyi Luciferase In a typical purification using a column with a 30 ml bed volume, the column is preequilibrated with about 100 ml of sample application buffer (used in the final dialysis of the sample) at a flowrate of 40 ml/hr maintained with a peristaltic pump. A typical sample would have an absorbance of 40-50 at 280 nm and 25,000 to 50,000 LU/ml luciferase activity (assayed with n-decanal; 1 L U (light unit) = 9.8 × 109 quanta/sec). The centrifuged (to remove material precipitating during dialysis) sample is applied to the column through the peristaltic pump at a flow rate of about 40 ml/hr. Fractions are collected from the effluent and assayed for protein (absorbance at 280 nm) and for luciferase. When the luciferase activity in the effluent reaches about 5-10% of the activity in the sample, application is terminated. A typical column of 30 ml bed volume can accommodate about 4.0 × 10 6 total light units, and loading of the sample usually requires between 1.5 and 2 hr. After sample loading, the column is washed with the same application buffer with which it had been equilibrated, also at about 40 ml/hr, until the absorbance and luciferase activity have returned to near-baseline values, usually about 4 hr. Luciferase elution is then effected by application of the elution buffer (see the table). The luciferase is eluted at a slower flow rate, about 5-10 ml/hr, to maintain a sharp elution profile. The luciferase elutes from the column in a sharp peak centered about 2-3 column volumes from the initiation of application of the elution buffer. Elution of the luciferase requires about 10-15 hr. While the elution can be carried out much more rapidly, both yields and purity suffer. By the procedure described here, we routinely achieve, by conservative pooling of fractions from the column, luciferase from V. harveyi of about 95% purity with overall yields of 50-75%. Affinity Purification of V. fischeri Luciferase The affinity method works well for both large scale and small scale purifications. To demonstrate the point, we will describe the purification
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P U R I F I C A T I O N OF B A C T E R I A L L U C I F E R A S E
107
of luciferase from 5 g of cell paste on a 1 ml column of D~bPA-Sepharose 6B. The column was preequilibrated as fast as allowed by the gel bed, about 50 ml/hr. The dialyzed sample from the 35-80% ammonium sulfate fractionation (13 ml with absorbance at 280 nm of about 40 and luciferase activity of 25,000 LU/ml assayed with n-dodecanal) was applied to the column in 1-ml aliquots and allowed to enter the bed as fast as possible. The luciferase activity and 280 nm absorbance of the effluent from the column were determined for each addition. The luciferase activity per unit volume in the effluent after the final addition was about 5% that of the original sample. The gel bed was then washed with seven 1.0-ml aliquots of application buffer, after which the absorbance at 280 nm and luciferase activity in the effluent were both near baseline. The luciferase was e|uted from the column by successive additions of 1.0-ml aliquots of elution buffer (see the table). Over half of the luciferase activity eluted in the second fraction, which by electrophoresis was estimated to be greater than 95% pure. By this small scale approach, the entire affinity purification requires less than 2 hr. The luciferase from P. phosphoreurn is readily purified by the same approach. The comparative instability of the luciferase from P. phosphoreum requires that care be taken in addition of fresh DTE to all buffers and that the eluent from the column be mixed immediately with ! M phosphate, pH 7.0. Otherwise, low yields are routinely observed. Use of D~bPA-Sepharose in a Batch Mode For very large scale rapid purifications of bacterial luciferase with yields that suffer only slightly compared with the ion exchange or affinity column methods, a batch affinity method may be employed. The sample preparation for the batch and column methods is identical. The affinity matrix is equilibrated with the application buffer (again, that used for the final dialysis) on a coarse sintered glass funnel. The buffer is added to the gel in aliquots and then suction dried by aspirator vacuum. The suction is turned off after each drying step, more buffer added and allowed to drip through, and then dried again by application of the vacuum. Equilibration of 500 g (suction-dried weight) of the gel requires about 1 liter of buffer. The suction dried equilibrated D~bPA-Sepharose on the sintered glass funnel is then titrated with the dialyzed sample after ammonium sulfate fractionation of the lysate obtained from about 500 g of V. harveyi cells. The sample volume from 500 g of cells is usually about 500-600 ml; aliquots of 100 ml are added to the gel and allowed to drip through. The gel is then dried after each addition by a short application of the aspirator vacuum. The luciferase activity is determined from the eluate after each
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addition, and the gel is considered to be saturated when the activity per unit volume in the eluate reaches about 5% that of the original sample. The capacity of the gel for pure luciferase from V. harveyi is about 8 mg/ ml gel; the capacity is about 2 mg/ml gel for luciferase that has been partially purified by ammonium sulfate fractionation. The luciferase content of 500 g of V. harveyi cells is about 1 g. After sample application is complete, the gel is washed on the sintered glass funnel with four to five 100-ml aliquots of the application buffer. The absorbance at 280 nm and the luciferase activity in the eluate are determined after each addition. The wash steps are performed as before; the buffer is applied without vacuum to the gel, allowed to drip through, and the gel is dried by a short application of the aspirator vacuum. When the absorbance at 280 nm and the luciferase activity in the eluate are low (A280 < 0.2), the luciferase is eluted by successive additions of the elution buffer (see the table). Elution usually requires seven 100-ml aliquots of buffer. Before each addition, 10 ml of 1.0 M phosphate, pH 7.0, is added to the vacuum flask so that the pH of the eluate will drop to near neutrality and the luciferase will be stabilized by the phosphate as it elutes from the gel. The elution buffer is added to the gel and allowed to drip through before application of the aspirator. Following drying of the gel after each 100-ml aliquot of elution buffer, the funnel is transferred to a fresh vacuum flask containing 10 ml of 1.0 M phosphate, pH 7.0. Each 110 ml fraction is assayed for luciferase activity and the absorbance at 280 nm is determined. Aliquots with the highest specific activity are pooled, concentrated by precipitation with ammonium sulfate at 80% saturation, and either dialyzed for experimental use or frozen as an ammonium sulfate paste. The batch method routinely yields about 60% of the luciferase in the dialyzed sample at a purity level of better than 90%. The overall batch method requires only about 3 hr, and from 500 g of V. harveyi cell paste, a yield of about 600-700 mg of luciferase is obtained. Acknowledgments This research was supported by grants from the National Science Foundation (PCM 8208589), the National Institutes of Health (AG 03697), the Robert A. Welch Foundation (A-865), the Upjohn Company, and a Hatch Grant (RI 6545).