Articles Anal. Chem. 1995, 67, 1301-1306
Preparation of Biotinylated P=Galactosidase Conjugates for Competitive Binding Assays by Posttranslational Modification of Recombinant Proteins Allan WRkowrki,t Mark S. Kine,* Sylvia Daunert,* and Leonidas 0. Backs* Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky 40506-0055
A biotinylated/I-galactosidaseconjugate prepared by the posttranslational modification of a recombinant fusion protein was used in the development of a heterogeneous binding assay for biotin. This conjugate was biotinylated at a predetermined location on a polypeptide tag attached to the N-terminus of B-galactosidase. A kinetic study using the purified conjugate showed that the genetically engineered biotinylated B-galactosidase has a slightly smaller Km for o-nitrophenyl B-D-galactopyranosidethan that found for the native enzyme. The biotinylated B-galactosidase was used to develop heterogeneous binding assaysfor biotin using both avidin and streptavidin-coated beads. Dose-response curves obtained by employingtwo merent batches of biotinylated B-galactosidaseprepared 4 weeks apart were essentially identical, indicating the potential advantage of long-term assay reproducibility attainable through the use of recombinant enzymeanalyte conjugates. ' h i s is made possible by the inherent specificity of the process of recombinant protein expression and posttranslational modification in Escherichia coli, resulting in the highly reproducible preparation of conjugates. Binding assays, such as homogeneous immunoassays, enzyme linked immunosorbent assays @USAS),and biotin-avidin binding assays, are widely used analytical techniquesfor the determination of biomolecules.1.2 Their popularity arises from their combined sensitivity and selectivity. Although numerous functional assays ' Present address: BAS Analytics, 1205 Kent Ave., West Lafayette, IN 47906.
* Department of Biochemistry, University of Kentucky, Lexington, KY 405360084. (1) Gosling, J. P. Clin. Chem. 1990, 36, 1408-1427. (2) Wilcheck, M.; Bayer, E. A. Methods Enymol. 1990, 184, 14-45. 0003-2700/95/0367-1301$9.00/0 0 1995 American Chemical Society
have been developed, most of the improvements in designing these techniques have concentrated on developing new labels' (enzymes, fluorophores, etc.) or enhancing the preparation of the binder used (antibodies, binding proteins, etc.), particularly with regard to monoclonal antibody prod~ction.3~~ Over the past few years, we have approached the area of binding assay development with the aim of understanding the factors influencing the fundamental analytical characteristics of these methods, such as reproducibility, limit of detection, and ~ensitivity.~-'~ This work has demonstrated that analyte-binder interactions greatly impact assay response, especially with regard to heterogeneity and degree of substitution of the analyte-label conjugate. In order to address these issues and prepare conjugates in a controlled fashion, we have recently used genetic engineering to produce peptide-enzyme conjugates for use in an ELISA for a peptides5 The application of recombinant DNA technology allowed a homogeneous population of monosubstituted peptide-enzyme conjugates to be prepared, and the ELISA developed using these genetically engineered conjugates was much superior to one in which the traditional activated ester (3) Nakamura, R M. In Immunochemical Assays and Biosensor Technology for the 1990's; Nakamura, R M., Kasahara, Y., Rechnitz, G. A, Eds.; ASM: Washington, DC, 1992; pp 205-227. (4) McCormack, R T.; Ludwig, J. R; Wolfert, R L. In Immunochemical Assays and Biosensor Technologv for the 1990s; Nakamura, R M., Kasahara, Y., Rechnitz, G . A, Eds.; ASM: Washington, DC, 1992; pp 57-82. (5) Witkowski, A; Daunert, S.; Kindy, M. S.; Bachas, L. G. Anal. Chem. 1993. 65, 1147-1151. (6) Barbarakis, M. S.; Qaisi,W. G.; Daunert, S.; Bachas, L.G. Anal. Chem. 1993, 65, 457-460. (7) Peak, S.-H.; Bachas, L. G.; Schra", W. Ana[. Biochem. 1993,210,145154. (8) Barbarakis, M. S.; Daunert, S.; Bachas, L. G. Bioconjwgate Chem. 1992,3, 225-229. (9) MacLean, A I.; Bachas, L G. Anal. Biochem. 1991, 195, 303-307. (10) Bachas, L. G.; Meyerhoff, M. E. Anal. Biochem. 1986, 156, 223-238.
Analytical Chemisfry, Vol. 67, No. 8,April 15, 1995 1301
coupling approach was used. However, since DNA only encodes for amino acids, the use of molecular biology to directly prepare enzyme-labeled analytes is limited to peptide-type analytes. In an attempt to extend the analytical benefits of the recombinant DNA approach of conjugate preparation to conjugates in which the analyte is not a peptide or protein, the use of posttranslational modification was investigated. Numerous examples of nonprotein cofactors that are covalently attached to proteins can be found in living systems, including lipoic acid in pyruvate dehydrogenasell and the biotin moieties in pyruvate carboxylaseI2 and trans~arboxylase.’~These prosthetic groups are attached to the proteins through posttranslational modification of the enzymes. In this process, the protein encoded by the gene is translated into an apoprotein, and a specific portion of this apoprotein acts as a recognition site for a ligating enzyme. Then, the ligating enzyme covalently attaches the cofactor to the apoprotein a t a precise location to give the active enzyme. If the recognition site of the ligating enzyme is known, it could be incorporated into a recombinant enzyme in order to direct the attachment of nonprotein analytes to a specific location on the recombinant enzyme. To test the feasibility of using posttranslational modilication to prepare genetically engineered enzyme-analyte conjugates for use in binding assays, we selected biotin as our model analyte. Biotin was chosen because of the widespread use of biotin-avidinbased assays in bioanalysis. Moreover, the preparation of biotinylatea recombinant proteins via posttranslational modification has been reported as a means to label, purify, and study protein^.'^ The basis of this approach is simply the construction of a piece of DNA that encodes the enzyme of interest attached to a peptide recognition sequence, as shown in Figure 1. The recognition peptide used here is that of biotin holoenzyme ligase (EC 6.3.4.9), an enzyme found in Escherichia coli. This enzyme attaches covalently a single biotin on the Gterminal side of its recognition site.13J4Thus, bacteria carrying the modilied DNA will produce the peptide-tagged enzyme, and the bacteria’s native biotin ligase will then biotinylate the peptide-tagged enzyme at a specific location. These enzyme-biotin conjugates can then be used as the labeled analyte to develop a binding assay for biotin. Because conjugate preparation via recombinant protein expression and posttranslational modifcation is highly reproducible, the use of recombinant enzyme-analyte conjugates also has the potential for enhancing long-term assay reproducibility. We now describe heterogeneous binding assays for biotin using avidin or streptavidin immobilized on beads and a p-galactosidase fusion protein, which has been biotinylated via posttranslational modification. EXPERIMENTAL SECTION Reagents. The Pinpoint Xa-Bgal expression vector and the Pinpoint protein purification system, containing expression vectors and Softlink soft release monomeric avidin resin, were obtained from Promega (Madison,wr). Tris(hydroxymethyl)aminomethane, free base (“ris) was from Research Organics (Cleveland, OH). Streptavidin immobilized on beads was obtained from Molecular Probes (Eugene, OR), and a broad molecular weight marker for (11) White, R H.; Bleile, D. M.; Reed, L J. Biochem. Biophys. Res. Commun. 1980,94, 78-84. (12) Moss, J.; Lane, M. N. Adu. Enymol. 1971,35,321-342. 10327-10333. (13) Cronan, Jr., J. E.;]. Biol. Chem. 1990,265, (14) Samols, D.; Thorton, C. G.; Marlii,V. C.; Kumar, G. IC;Haase, F. C.; Wood, H. G. J. Bid. Chem. 1988,263, 6461-6464.
1302 Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
DNA codina a biotin ligase reiognition sequence plasmid
w
1
Protein Translation
biotin tigare recognition sequence
’
‘
enzyme
I
Posttranslatlonal Modification by Biotin Ligase
Blotinylated Recombinant Enzyme Flgure 1. Schematic representationof the bacterial preparation of an enzyme-biotin conjugate by the posttranslationalmodification of a recombinant enzyme.
SDS-PAGE was purchased from Bio-Rad (Richmond, CA). The Ma-Bertani liquid medium (LB) and isopropyl thic+galactoside (IPTG) were purchased from GibceBRI, (Gaithersburg, MD) .The BCA protein assay reagent and avidin immobilized on beads were obtained from Pierce (Rockford, IL). 3-[4Methoxyspiro(l,2dioxetane-3,2’-tricyclo13.3.1.lW7Idecan)-4yllphenylp-Dgalactopyranoside (AMPGD) and Emerald luminescence amplifying reagent were purchased from Tropix (Bedford, MA). Bovine serum albumin @SA),ampicillin (amp), lysozyme, phenylmethylsulfonyl fluoride, the sodium salt of deoxycholic acid (DOC), biotin, Sephadex G150 (120-i4 mesh) size-exclusion gel, o-nitrophenyl p-Dgalactopyranoside (ONPG), and all other reagents employed were from Sigma (St. Louis, MO). All chemicals were of reagent grade or better and were used as received. All solutions were prepared using deionized (Milli-Q water purification system, Millipore, Bedford, MA) distilled water. Apparabs. Bioluminescence measurements were made on an Optocomp I luminometer obtained through GEM Biomedical (Carrboro, NC) using a total count time of 3 s and dual 1O@pL fixed injectors. All experiments were conducted at room temperature. When the ONPG substrate was used, visible absorbance measurements were performed at 405 nm on a SYVA SI11 spectrophotometer interfaced with an Emit CP-5000 clinical processor (SYVA, Palo Alto, CA). Preparation and Isolation of BiotinyIated p-Galactosidase. Bacteria (E. coli strain JM109) were transformed with the Pinpoint Xa-pgal expression vector, a DNA plasmid encoding for B-galactosidase expressed as a fusion protein with a naturally biotinylated polypeptide tag at the N-terminus of the enzyme. All molecular biology procedures were performed using standard protocol^.'^ The transformed bacteria were cultured to express the fusion protein according to the manufacturer‘s instructions. The bioti(15) Maniatis, T.;Fritsch, E. F.; Sambrook, J. Molecular Cloning: A Luboratoy Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1982.
nylated #?-galactosidase(BLAC) fusion protein was isolated from the bacteria by cell lysis using a lysozyme/DOC/sonication protocol. The entire isolation process was conducted at 4 "C. The harvested cells were resuspended by adding 30 mL of lysis buffer (50 mM NaCl and 5% (vlv) glycerol in 50 mM Tris-HC1, pH 7.5) and stimng for 5 min. Next, 30 mg of lysozyme and 180 pL of a solution containing 44 mg of phenylmethylsulfonyl fluoride in 1 mL of 95%ethanol were added to the resuspended cells and stirred for 20 min. To this mixture was added 30 mg of the sodium salt of deoxycholic acid and the contents were stirred for an additional 5 min. The lysis solution was then split into two 15mL fractions and transferred to 30-mL Corex tubes for sonication. These tubes containing the cell extracts were sonicated on ice using 15s pulses with a 15srest between pulses. The total sonication time for each tube containing the cell extracts was 5 min. The crude lysate was then centrifuged at 10 000s for 15 min at 4 "C to remove any remaining cellular debris, and the supernatants were recombined in a clean tube. This crude BLAC solution was stored at 4 "C until further use. Pwikation of the Biotinylated /?-Galactosidase Fusion Protein. Two purification protocols were developed for use with the BLAC fusion protein. The first method involved column chromatography using the Softlink monomeric avidin resin provided by Promega. The resin was preconditioned according to the manufacturer's instructions and packed into a 5mL disposable chromatography column. The beads were equilibrated by washing four times with 3 mL of column buffer (same composition as lysis buffer above), loaded with crude cell lysate, rinsed, and eluted with elution buffer (column buffer containing 5 mM biotin). The eluted fractions containing the highest levels of P-galactosidase activity were pooled and placed in standard cellulose dialysis tubing (Spectrum Medical Industries, Los Angeles, CA) with a molecular weight cutoff of 12 000-14 000. The affinity-purified BLAC was then dialyzed against three changes of 2 L of 1.0 mM magnesium chloride in 0.100 M sodium phosphate, pH 7.0, to remove any traces of free biotin. Each dialysis step was 8 h long. The second purification procedure was a batch method designed to speed up the affinity chromatography step and performed in conjunction with a size-exclusion column to remove the large excess of lysozyme added during cell lysis. The Softlink resin was regenerated according to instructions supplied and divided into two 15mL centrifuge tubes. Each tube was washed three times with column buffer, and then 10 mL of crude lysate was added to each tube. The tubes were mixed by inversion at 4 "C for 4 h and centrifuged to pellet the resin. Next, the resin was rinsed and the fusion protein was released by an overnight incubation in elution buffer according to the manufacturer's protocol. The resin was centrifuged,and the released BLAC was collected in the supernatant. Following this batch purification, the BLAC fusion protein was passed over a Sephadex G150 (120-A mesh) size-exclusion column to remove any biotin or lysozyme present. A 4mL aliquot of the batch-purified BLAC was loaded onto the sizeexclusioncolumn by gravity flow. The BLAC protein was then eluted using column buffer as the mobile phase under gravity flow; the presence of protein was monitored by absorbance at 280 nm. Thirty fractions of 1mL each were collected beginning 30 min after elution was started. The eluted fractions containing signiiicant BLAC activity were pooled and dialyzed against 2 L of magnesium chloride/phosphate buffer as above. The purified
BLAC was aliquoted into 1-mL tubes and stored at -20 "C. It should be noted that equivalent results were obtained in all assays using BLAC purified by either protocol. SDS-PAGE of Biotinylated /?-Galactosidase. In order to monitor the purification of the BLAC fusion protein, samples at various stages in the purification protocol were analyzed via a SDS-PAGE minigel using a Mini-Protean I1 apparatus from BioRad. The running part of the gel was a 5-20% acrylamide gradient, and this was used in conjunction with a 3%acrylamide stacking gel. The protein bands were visualized using Coomassie Blue. The protein size for each band was determined by comparison to a broad molecular weight marker run adjacent to the other samples. The bands in the molecular weight standard for M W 14 400 and 116 300 correspond to hen egg white lysozyme and to one subunit of E. coli #?-galactosidase,respectively. Assay for Biotinylated /?-GalactosidaseActivity. In order to determine the lowest amount of BLAC that was feasible to use, an activity assay was performed using the purified BLAC. An assay buffer containing 1.0 m M magnesium chloride and 0.10% BSA in 0.100 M sodium phosphate, pH 7.0, was prepared (assay buffer). The BLAC substrate chosen was AMPGD, a chemiluminescent substrate for #?-galactosidase.The AMPGD was used in conjunction with Emerald luminescence amplifier as previously reported.16 Specifically, a substrate solution was prepared fresh daily by adding 16.5 pL of AMPGD concentrate to 10.0 mL of a buffer containing 1.0 mM magnesium chloride in 0.100 M sodium phosphate, pH 7.0. The luminescence of the product was triggered by the injection of an enhancer solution, which was 0.20 M sodium hydroxide containing 10%(v/v) Emerald luminescence amplifier. Serial dilutions of the purified stock BLAC solution were prepared using assay buffer. (The substrate solution and all BIAC dilutions were kept on ice to prevent loss of activity.) To prepare calibration curves, 250 pL of assay buffer, 200 pL of substrate solution, and 40 pL of varying concentrations of BLAC were combined in 12 x 75 mm glass tubes. After incubation with shaking for 30 min, the chemiluminescence of the enzymatic product was measured by placing each tube in an Optocomp I luminometer and injecting a total of 200 pL of enhancer solution. The luminometer was programmed to have a 0.5s delay between each 1WpL injection and a 2-s delay after the last injection before data were collected. The light intensity was measured over a 5 s interval. All luminescence intensities reported are the average of at least three replicate samples and have been corrected for the contribution of the blank. Kinetic Study of Biotinylated /?-Galactosidase. In order to compare the enzymatic activity of the BLAC fusion protein to that of native ,&galactosidase, a kinetic study was performed using ONPG as the substrate. For each experiment,2.85 mL of ONPG and 150pL of an enzyme solution were combined in a glass tube, the tube was mixed rapidly by inversion, and the sample was aspirated into a SYVA spectrophotometer for absorbance measurements at 405 nm. After an initial delay of 15 s, the solution absorbance was recorded at lGs intervals over a period of 5 min. The total change in absorbance over this period ( A N A t ) was then used to calculate the initial velocity (v) of the reaction based on the volume of the cell and the light path length using a molar absorptivity of 3.5 x M-l cm-I for ONPG.I7 AU kinetic studies (16) Jain, V. IC; Magrath, I. T. Anal. Biochem. 1991,199, 119-124. (17) Biochimica Information; Keesey, E. J., Ed.; Boehringer Mannheim Biochemicals: Indianapolis, IN, 1987; pp 167-170.
Analytical Chemistry, V d . 67, No. 8, April 15, 1995
1303
develop improved binding assays, as in the case of a peptide ELI% using a peptide-enzyme f u ~ i o n .In ~ that earlier report, genetic engineering of the enzyme-labeled analyte allowed for the exact control of the reagent composition, resulting in conjugates that performed significantly better in the peptide assay than ones prepared by conventional methods. However, only protein or peptide analytes can be determined using enzyme-analyte fusion proteins. In an attempt to extend the benefits of using recombinant DNA technology to develop assays for non-peptide analytes, a new methodology was considered. By constructing a fusion protein based on an enzyme label connected to the recognition sequence of a speciiic protein-modifying enzyme, genetically engineered conjugates with nonpeptide analytes can be prepared in E. coli. We now demonstrate the feasibility of this approach using a biotinylated /?-galactosidaseprepared by posttranslational modification of a recombinant protein to develop a heterogeneous binding assay for biotin. As shown schematically in Figure 1,a biotin-/?-galactosidase conjugate (BLAC) was prepared by coupling the polypeptide recognition sequence of the enzyme biotin ligase to the N-terminus of /?-galactosidase. The DNA encoding this fusion protein can be readily obtained using the Pinpoint Xa protein expression system, which makes use of DNA plasmids containing the nucleotide base pairs encoding the biotin ligase recognition sequence adjacent to a multiple cloning site. The multiple cloning site is a specially designed series of nucleotides that can be speciiically cleaved by restriction enzymes, thus allowing the insertion of the gene for the enzyme after the DNA for the biotin ligase recognition sequence. Once this fused DNA is translated, the native bacterial enzyme biotin ligase attaches a single biotin at a precise location on the recognition sequence.13J4 Following the induction of the bacteria to synthesize the fusion protein, the BLAC was isolated and purified as described in the Experimental Section. The purification process was monitored using a standard SDS-PAGE minigel. As can be seen in Figure 2 (lane 2), affinity purification using the monomeric avidin alone resulted in the isolation of two predominant bands, one corresponding to BLAC and the other to lysozyme. The lysozyme contamination occurred because of the large quantity of lysozyme used during the isolation protocol. Although subsequent data indicated that the presence of lysozyme had no effect on the biotin assay developed, a highly purified BLAC solution was desired for our studies. Therefore, the monomeric avidin-purifiedBLAC was passed through a size-exclusion chromatography (SEC) column RESULTS AND DISCUSSION to remove the lysozyme (see Figure 2, lane 1). Thus, the Recently, several groups have utiliied techniques employed combined af&inity/SEC purification of BLAC resulted in a homoin molecular biology to prepare novel binding a s s a y ~ . ~ J ~ - geneous ~~ recombinant product that is signiiicantly cleaner than Recombinant fusion proteins have been employed successfully to commercially available /?-galactosidase (see Figure 2, lane 5). After obtaining the puritied fusion protein, our initial step was (18) Lindbladh, C.; Person, M.; Bulow, L.; Stahl, S.; Mosbach, K. Biochem. to determine whether the addition of the biotinylation peptide tag Biophys. Res. Commun. 1987,149, 607-614. (19) Hunger, H. D.; Flachmeier, C.; Schmidt, G.; Behrendt, G.; Coutelle, C. Anal. and subsequent posttranslational moditication affected the enzyBiochem. 1990,187, 89-93. matic activity of the /?-galactosidase. Two criteria were used to (20) Brandes, W.; Maschke, H. E.; Scheper, T. Anal. Chem. 1993,65, 3368determine this: (a) the limit of detection of the enzyme and (b) 3371. (21) Henderson, D. R; Friedman, S. B.; Hams, J. D.; Manning, W. B.; Zoccoli, the enzyme kinetics. First, a chemiluminescent assay for enzyM. A Clin. Chem. 1986,32, 1637-1641. matic activity using AMPGD was employed to determine the (22) Engel, W. D.; Khanna, P. L. 1.Immunol. Methods 1992,150, 99-102. lowest amount of BLAC and native /?-galactosidasethat could be (23) Kobatake, E.; Nishmori, Y.; Ikariyama, I.; Aizawa, M.; Kato, S.Anal. Biochem. 1990,186, 14-18. detected under the conditions of our assay. The limits of detection (24) Gillet, D.; E m , E.; Ducancel, F.; Gaillard, C.; Ardouin, T.; Istin, M.; Menez, found here for BLAC and native /?-galactosidaseare 2.0(f0.1) and A; Boulain, J.-C.; Grognet, J. M. Anal. Chem. 1993,65, 1779-1784. 1.6(f0.1) pg, respectively. This corresponds to a detection limit (25)Stirling, D. A.; Petrie, A; F'ulford. D. J.; Paterson. D. T. W.; Stark, M. J. R. Mol. Microbial. 1992,6, 703-713. of 3.9(f0.2) amol for BLAC and 3.0(f0.2) am01 for the native were performed at 20 "C. The determination of initial velocity was performed in triplicate at each substrate concentration using both BLAC and native /?-galactosidase,and the data were used to construct Lineweaver-Burk plots of l / v vs l/[substratel. Binder Dilution Studies. In order to determine how much of the avidin- or streptavidincoated beads were necessary to use for each binding assay, binder dilution studies were conducted. Streptavidin or avidin beads were transferred to separate centrifuge tubes and rinsed once with 15 mL of assay buffer, followed by centrifuging and decanting the supernatant. The rinsed beads were then diluted with an equal volume of assay buffer to yield a stock suspension of each bead type. Serial dilutions of the beads were made using assay buffer. By using different volumes of these suspensions, a range of bead quantities was tested. The desired bead volumes were pipeted into glass tubes, and the total volume was brought to 500 pL with assay buffer. Next, 40 pL of a 2.1 x M solution of BLAC in assay buffer was added to each tube, and the mixture was allowed to incubate for 30 min with shaking. The tubes were then centrifuged, the supernatant was decanted, and the tubes were rinsed three times with 3 mL of assay buffer. After the final rinse, most of the supernatant was removed, with 0.25 mL of solution remaining in each tube. To determine the amount of conjugate bound to the solid phase, 200 pL of AMPGD substrate solution was added to each tube, which were incubated for 30 min with shaking. The luminescence of the enzymatic product was measured using the luminometer and enhancing solution as described above. All luminescence intensities reported are the average of at least three replicate samples. Dose-Response Curves. Dose-response curves were constructed by incubating 100 pL of various concentrationsof biotin with a fixed amount of bindercoated beads and BLAC. Serial dilutions of biotin were made from a 2 x M stock solution by diluting with assay buffer. For each assay, 300 p L of assay buffer, 100 pL of bindercoated bead suspension, and 100 pL of varying concentrations of biotin were combined in a glass tube and incubated with shaking for 30 min. The 100 pL of bindercoated beads was delivered from the stock suspension of avidin beads or a 1:lO dilution of the streptavidin beads. After the initial incubation period, 40 pL of a 2.1 x M BLAC solution (44pg of BLAC) was added and the mixture was allowed to incubate with shaking for an additional 30 min. This was followed by the rinsing, substrate reaction, and product quantification steps as described above.
1304 Analytical Chemistry, Vol. 67, No. 8,April 15, 7995
M. W. =
200,000
--
1 16 , 3 0 0 97,400 66,200
-
45,000
-
31,000
--
21,500 14,400 6,500
1
2
3
4
5
6
Figure 2. SDS-PAGE minigel of purified BLAC. From left to right, lanes 1-6 contain BLAC after consecutive affinity chromatography and size-exclusion chromatography steps (1), BLAC after monomeric avidin affinity chromatography (2),crude BLAC lysate (3),lysozyme (4), commercially available /?-galactosidase( 5 ) , and broad-range molecular weight markers (6). The molecular weights of the marker bands are shown in the column on the right.
P-galactosidase. The definition of detection limit used is the amount of enzyme necessary to produce a luminescent signal that is 2-fold greater than the noise in the background signal. Because it appeared from the limit of detection data that the BLAC was not as enzymaticallyactive as the native @galactosidase, a kinetic study was performed to determine the Michaelis constant (K,J for BLAC. The magnitude of the K m can be taken as a measure of the apparent affinity of the fusion protein for a /%galactosidase substrate, with a larger K m corresponding to weaker substrate affinity and vice Standard LineweaverBurk doublereciprocal plots were constructed using either BLAC or native /?-galactosidasewith the substrate ONPG. The assays were conducted using dilutions of enzyme, which delivered 1pg of BLAC or native /3-galactosidase per tube. These graphs were linear, and under the conditions of our assay, the Km’S were determined to be 0.128(f0.003) mM for BLAC and 0.175(f0.005) mM for fiative /3-galactosidase. These values are similar to the Km of 0.11 mM reported for ONPG by Tenu et al. at the same pH and similar ionic strength.n Thus, it appears that the BLAC fusion protein has a slightly greater affinity for ONPG than does the native enzyme. On the basis of graphical analysis of the kinetic data, the maximum enzymatic velocity (Vmd was also calculated for both enzymes used. The V m x for BLAC using ONPG was found to be 7.58(f0.02) pM/min, while that of the native /?-galactosidasewas ll.ZO(f0.02) pM/min under our assay conditions. When corrected for the differences in molecular weights of these two proteins (due to the addition of the biotin ligase amino acid recognition sequence), the molar activity (turnover number) for BLAC is approximately 64% of that of the native P-galactosidase. We attribute the decrease in molar activity of BLAC to structural changes in the fusion protein that alter the substrate affinity and in turn reduce the turnover rate of the enzyme. This assumption is also in agreement with the relative limits of detection of BLAC and native /?-galactosidaseas determined above. (26) %gel, I. H. Biochemical Calculations; 2nd ed.;John Wiley and Sons: New York, 1976. (27) Tenu, J.-P.; Viratelle, 0. M.; Gamier, J.; Yon, J. Eur. 1.Biochem. 1971,20. 363-370.
6000 3
i;:
5000
5
4000
v)
c
-2c
3000 2000 0
2 4 6 8 10 12 Volume Streptavidin Beads, pL
Figure 3. Binder dilution curve obtained using 44 pg of BLAC and different amounts of streptavidin-coatedbeads. The y-axis represents the luminescence intensity of the enzymatic product measured in relative light units (RLU).
In order to develop the heterogeneous biotin assay, it was necessary to determine the optimum amount of bindercoated beads to use in each assay. This was achieved by constructing a binder dilution curve using streptavidin immobilized on beads, and these results are depicted in Figure 3. In this study, 40 pL of a 2.1 x 10-I2 M solution of BLAC (corresponding to 44 pg of BLAC) was used to provide a fixed amount of conjugate as the bead volume was varied. Because the biotin-streptavidin binding constant is 1 x 1015M-I, regeneration of the solid phase was not attempted and the streptavidin beads were discarded after each use. The BLAC fusion protein was employed in the development of a heterogeneous binding assay for biotin, and a dose-response curve for this vitamin was constructed. Figure 4 shows the effect on the amount of solid-phase enzymatic activity detected when varying amounts of biotin analyte are incubated in the presence of a fixed amount (44 pg) of BLAC. This assay was performed in a sequential manner, with biotin being allowed to incubate with 10pL of streptavidin beads (delivered in 100-pLaliquots of a 1:lO Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
1305
I
'I"
T
0 -9
I
-7
-8
1
.
-6
1
-5
'
-4
log [Biotin, M I
log [Biotin, MI Figure 4. Dose-response curves for biotin generated by incubating 44 pg of BLAC and varying concentrations of biotin in the presence of a fixed amount of binder-coatedbeads: (X) 1OOpL of avidin beads; (0)10 pL of streptavidin beads. The y-axis representsthe percentage of change in luminescence intensity relative to the intensity at the highest biotin concentration.
Figure 5. Dose-response curves for biotin generated with streptavidin beads under identical conditions using different batches of BLAC: (0) affinity-purified BLAC; (X) affinity/SEC-purified BLAC prepared 4 weeks later. The y-axis represents the percentage of change in luminescence intensity relative to the intensity at the highest biotin concentration.
bead dilution) for 30 min, followed by the addition of the BLAC protein. As expected, high biotin concentrations essentially saturate the binding sites on the solid phase, resulting in low enzymatic activity being detected on the beads (Figure 4). However, at lower biotin concentrations, the majority of the streptavidin sites are occupied by the BLAC conjugate and the luminescence intensity from AMPGD consumption is higher. The limit of detection in this assay is 6 x M biotin, based on twice the noise in the signal at the lowest biotin concentrations. The data obtained, although not necessarily indicating the most sensitive assay for biotin reported, demonstrate the feasibility of using posttranslational modifcation to prepare labeled analytes for use in binding assay development. Since avidin can be used in place of streptavidin to develop biotin assays, avidin immobilized on beads were also evaluated for use with BIAC. A binder dilution curve was constructed (data not shown) indicating an optimal avidin bead volume of 100 pL for each assay. A new dose-response curve was prepared using the above quantity of avidin beads, varying concentrations of biotin, and 44 pg of BLAC. The avidin-based heterogeneous dose-response curve is shown in Figure 4 as a direct comparison to the similar curve obtained using streptavidin beads. The avidin beads yield a dose-response curve that is shifted toward slightly worse detection l i i t s . This is expected since more binder is being used in the case of avidin. The detection limit for the avidinbased assay is 8 x lo-' M biotin. In an earlier paper, we indicated that one of the primary advantages of the use of recombinant conjugates in binding assays should be an increase in assay repr~ducibility.~ Because the same protein is encoded by the fusion gene each time it is translated, there should be no lot-to-lot variations in conjugate composition or activity. This would be a significant improvement in assay development, since variations in conventional conjugate preparation generally require the assay to be reoptimized for each new batch of conjugate used. To experimentally verify the hypothesis that recombinant conjugates lead to enhanced assay reproduc-
ibility, two biotin dose-response curves were constructed using streptavidin beads under identical conditions. These curves are shown in Figure 5. The first curve (open circles) was prepared using BLAC purified only by the monomeric avidin column as described in the Experimental Section. The second curve (X's) was obtained using BLAC isolated 4 weeks later and purified using the combined affinity/SEC protocol. As is evident in Figure 5, the dose-response curves are nearly identical using the genetically engineered BIAC, regardless of puritication protocol or batch used. In conclusion, we have demonstrated that enzyme-analyte conjugates prepared in vivo by posttranslational modifcation of recombinant proteins can be used to develop binding assays for nonprotein analytes. This approach allows one to extend the advantages of the controlled production of a homogeneous population of monosubstituted conjugates attainable via genetic engineering to analytes not directly encoded by DNA Furthermore, dose-response curves prepared using batches of BLAC produced at different times and purified by different protocols were essentially identical, providing experimental evidence for the improved assay reproducibility that can be achieved through the use of recombinant enzyme-analyte conjugates in binding assays.
1306 Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
ACKNOWLEWMENT
This work was supported in part by the National Science Foundation (CTS9307518). AW. gratefully acknowledges the support of a Dissertation Year Fellowship from the University of Kentucky. The authors also thank Rick Salatino of Promega for supplying the Pinpoint %-#?gal expression vector, and Dr. Marcielle de Beer for technical assistance. Received for review September 12, 1994. Janurary 19, 1995.@
Accepted
AC940905T @
Abstract published in Advance ACS Abstracts, February 15, 1995.
