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Journal of Immunological Methods, 137 (1991) 199-207 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100110X
JIM05858
Preparation of a genetically fused protein A/luciferase conjugate for use in bioluminescent immunoassays. C h r i s t e r L i n d b l a d h , K l a u s M o s b a c h a n d L e i f Biilow Department of Pure and Applied Biochemistry, Chemical Center, Universityof Lund, P.O. Box 124, S-221 O0Lund, Sweden
(Received 22 August 1990, revised received 19 October 1990, accepted 19 November 1990)
The genes encoding staphylococcal protein A and bacterial luciferase (Vibrio harveyi) were fused in-frame in order to obtain a general marker enzyme for bioluminescent immunoassays. Two constructs were made where protein A was ligated to the first and the 12th amino acid residue, respectively, of the N terminus of the fl subunit of luciferase. Only the first fusion protein encoding the entire/3 subunit was able to form an enzymatically active luciferase complex when expressed together with the a subunit. The fusion of protein A to luciferase did not notably alter the emitted wavelength spectrum or its stability to urea treatment. The fusion protein was found to retain at least 50% of the specific bioluminescent activity compared to native luciferase. In preliminary tests, this hybrid protein was shown to be useful in bioluminescent immunoassays. Key words: Protein A; Luciferase; Bioluminescence; Immunoassay; Fusion protein
Introduction
The use of radioactive- or enzyme-labelled antigens or antibodies has long been one of the best tools in the detection and quantification of biological substances. Radioimmunoassay has, however, several practical drawbacks compared to enzyme-linked immunoassays including safety, labelling and stability of the radioactive compound. This has led biochemist to further improve the
Correspondence to: C. Lindbladh, Department of Pure and Appfied Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden. Abbreviations: LB-broth, Luria-Bertani broth; DTT, dithiothreitol; IgG, immunoglobulin G; kb, ldlobase pair; Xgal, 5-bromo-4-chloro-3-indolyl-fl-D-galactopyranoside;IPTG, isopropyl-fl-D-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulphate-polyacrylarnidegel electrophoresis.
sensitivity and applicability of several enzymes which are commonly used in the labelling of conjugates. Different techniques such as the use of substrates generating fluorescent products (Milby, 1985) or coupled enzyme systems with substrate recycling (Bates, 1987) have thus been employed. Much attention has also been given to detection systems based on luminescence. These methods include the use of enzyme labels such as luciferase, alkaline phosphatase and horseradish peroxidase which together with a suitable substrate generates light (McCapra, 1989). The latter two enzymes have been utilized extensively since enzyme conjugates can easily be prepared chemically. Luciferases are also attractive marker enzymes known to be very sensitive and convenient in the detection of various enzymes and metabolites. Both bacterial and firefly luciferases have been e m p l o y e d for such b i o a n a l y t i c a l p u r p o s e s
200
(Ugarova and Lebedeva, 1987; Wienhausen et al., 1987). In order to make an immunoassay based on luciferase as a marker enzyme it is desirable to attach luciferase onto an antigen or an antibody. However, the bacterial enzyme tends to lose much of its activity when treated with the chemical crosslinking agents used in these procedures, as can be seen for instance in the immobilization of luciferase to solid supports (Wienhausen and DeLuca, 1982). The problems associated with such chemical procedures can be circumvented by preparing an in-frame gene fusion between the protein of interest and bacterial luciferase (Vibrio harveyi). In this paper we describe the construction of such a protein A/luciferase hybrid protein. The gene product obtained is a well-defined bifunctional enzyme conjugate that can be used directly in a bioluminescent immunoassay. Protein A was chosen because of its ability to bind I g G from a number of different species (Langone, 1982) and thereby create a general marker enzyme conjugate, suitable for bioluminescent immunoassays. Previously, similar artificial hybrid enzymes, prepared by gene fusions, have found wide application in protein purification (Smith et al., 1984; Nilsson et al., 1985; Persson et al., 1988), sequentially operating enzyme systems (Billow et al., 1985; Ljungcrantz et al., 1989), protein stabilization (Guo et al., 1984) and in analytical enzyme immunoassays (Lindbladh et al., 1987). Bacterial luciferase is a heterodimeric enzyme with two different subunits, a and fl encoded by the lux A and lux B genes, which initially interact as partly folded structures before the final folding takes place (Waddle et al., 1987). Both genes have been cloned and sequenced (Cohn et al., 1985; Jonston et al., 1986). The active enzyme produces light when it reacts with 02, F M N H 2 and a longchain aldehyde according to the following reaction: F M N H 2 + R C H O + 0 2 ---* F M N + R C O O H + H 2 0 + light
Due to the instability of F M N H 2, it is convenient to generate this substrate from F M N by the enzymes diaphorase or N A D ( P ) : N A D ( P ) H oxidoreductase, which use N A D H for this purpose. It is generally accepted that the single active center of luciferase is located on the a subunit. The func-
tion of the fl subunit is unknown but it is necessary for the enzymatic activity of luciferase. Recently, it has been demonstrated that it is possible to fuse at least 17 amino acid residues to the N terminus of the a subunit of luciferase. This was done in order to investigate the feasibility of using the lux A gene of bacterial luciferase as a reporter gene for gene fusions (Olsson et al., 1988). Furthermore, an enzymatically active lux A / l u x B fusion has been prepared by site-directed mutagenesis (Boylan et al., 1989). In this report we wish to describe an extension of such hybrid luciferase proteins, exemplified by the construction of a biologically active in-frame fusion between protein A and the amino terminal end of the fl subunit of luciferase and its potential use in bioluminescent immunoassays. We also describe the construction of a vector suitable for various in-frame fusions with luciferase (lux B).
Materials and methods
Enzymes and chemicals Restriction endonucleases and T 4 - D N A ligase were purchased from Boehringer Mannheim, Germany, and mung bean nuclease was from Pharmacia-LKB, Sweden. All enzymatic reactions were carried out as recommended by the manufacturer. Xgal, IPTG, N A D H , F M N and diaphorase were obtained from Boehringer Mannheim. DEAE-Sepharose and Sepharose 4B were purchased from Pharmacia-LKB and n-decyl aldehyde was from Sigma, U.S.A. The rabbit I g G antibodies used in the immunoassay were obtained from Dakopatts, Denmark. All other chemicals were of analytical grade and commercially available. Bacterial strains and plasmids E. coli JM105 (Messing, 1983) and N 4830-1 (Gottesman et al., 1980) were used as recipients for bacterial transformation of plasmids. Plasmids pUC19, p R I T 2 T and the kanamycin resistance gene were purchased from P h a r m a c i a - L K B . Plasmids pJH2 and pJH5 coding for the luciferase genes lux A and lux B, respectively, have been described previously (Waddle et al., 1987). Oligonucleotide synthesis was performed on a
201 381A D N A Synthesizer, Applied Biosystems, using the phosphoramidite method (Beaucage and Caruthers, 1981) at the molecular biology core facility, University of Lund, Sweden. The oligonucleotides were purified on a M o n o Q column. Cells were grown in an LB medium (Miller, 1972) containing ampicillin and kanamycin at 100 # g / ml and 30 ~ g / m l , respectively. Xgal, 40 /~g/ml, and IPTG, 0.1 mM, were used when necessary.
Production and purification of protein A/luciferase conjugate The bacterial culture was grown in LB medium containing ampicillin and kanamycin at 33 ° C. At ODs50 = 0.5, the culture was induced by heating to 42 o C and I P T G was added. The fermentation was stopped when ODss 0 reached 2.0. After centrifugation at 6000 x g for 5 min, the cell pellet was washed once with buffer A (100 m M phosphate buffer p H 7.3, 1 m M D T T ) and finally suspended in the same buffer containing 1 m M E D T A and 0.5 m g / m l of lysozyme. The cells were then lysed by putting the suspension on a rocking table for 30 rain at 22 ° C followed by sonication for 3 x 60 s (output 5, Sonifer B-30, Branson Sonic Power). All subsequent operations were carried out at 4 ° C. The crude protein extract was clarified by centrifugation at 12,000 x g for 5 rain, and solid ammonium sulfate was added to the supernatant until 55% saturation was reached. After centrifugation at 12,000 x g for 5 rain the protein pellet was dissolved in buffer A and dialysed overnight against the same buffer. Any precipitate was removed by centrifugation. The protein extract was then applied to a column of DEAE-Sepharose previously equilibrated with the same buffer. The fusion protein was eluted at 0.1 M NaC1 in a linear gradient of 0-0.5 M NaC1. The conjugate was further purified on a Superose 12, gel filtration column, Pharmacia-LKB, when examining its specific activity. A similar purification protocol was used for the preparation of native luciferase obtained from growing cells harboring plasmids p J H 2 and pJH5. After purification, the hybrid protein was routinely stored at 4 ° C. 1 m M D T T was added weekly.
Identification of the fusion protein S D S - P A G E was performed on a 10% polyacrylamide slab gel using a Tris-HC1 p H 8.3, dis-
continuous buffer system according to Laemmli (1970). The M r of the fusion protein was determined by comparing the relative mobility with those of the standard calibration proteins obtained from Pharmacia-LKB. Western blotting was performed as recommended by the manufacturer, JKA-Biotech, Denmark, The antibodies used in the reaction were peroxidase-conjugated rabbit immunoglobulin (Dakopats). H202 and 4-chloro1-naphthol were employed as substrates to visualize the peroxidase. The protein concentration was determined according to Bradford (1976), using bovine serum albumin as the standard.
Bioluminescence measurements Luminescent activity in mV was measured in a total volume of 200 /~1 by mixing the fusion protein with 100 m M sodium phosphate buffer at p H 7.3, 0.9% ( w / v ) NaC1, 0.1% ( w / v ) gelatin, diaphorase 0.35 U, F M N 0.03 m M and 0.001% n-decyl aldehyde, previously dissolved in ethanol. The reaction was started by the addition of N A D H to a final concentration of 0.25 m M and the peak luminescent value was measured. Luminescence from protein A/luciferase bound to IgG-Sepharose was monitored in a similar way. Bioluminescence from whole cells was monitored by adding 10/~1 n-decyl aldehyde to 0.5 ml cell suspension followed by vigorous mixing. All reactions were carried out at 22 ° C in a luminometer, Wallac 1250, or a Hitachi F-3000 fluorescence spectrophotometer.
Immobilization of lgG on Sepharose 4B The immobilization of antibodies was performed according to K o h n and Wilchek (1982), with some minor modifications. 2 g of Sepharose 4B were washed with ice cold water followed by 30% acetone and 60% acetone. The Sepharose was then suspended in 60% acetone and chilled below 0 o C. 60 mg CNBr and 850 m m o l triethylamine were added to the suspension and allowed to react for 45 s. The reaction mixture was then immediately poured into ice cold acetone, 0.1 M HC1, 50% (v/v). The mixture was subsequently transferred back to water by washing with ice cold 60% acetone followed by 30% acetone and water. The activated agarose gel was suspended in 0.1 M N a H C O 3 at p H 8.4 and 100 /~1 rabbit I g G anti-
202
bodies (Dakopats) were allowed to react overnight at 4” C. After coupling, additional reaction sites on the Sepharose gel were quenched with 0.2 M ethanolamine in 0.2 M Tris-HCl pH 8.0 for 30 min at 0°C. The IgG-Sepharose mixture was washed thoroughly with storage buffer (100 mM phosphate buffer pH 7.3, 0.9% NaCl and 0.02% NaN,) and suspended in the same buffer to give a final concentration of 200 mg IgG-Sepharose gel/ml. Bioluminescent immunoassay procedure Different amounts of IgG-Sepharose gel were mixed with pure Sepharose gel to give the same amount of gel in each test tube (50 mg). Equal amounts of purified protein A/luciferase conjugate, having an activity corresponding to 15,000 mV on the luminometer, were added to each test tube and the mixture was incubated for 3 h at 22 o C in a total volume of 350 ~1. Before monitoring the bioluminescence, the Sepharose mixture was washed twice with 2 volumes of storage buffer.
pRIT2T
I
Eco
RI
mung
bean
nucleate
Barn
Pst
HI
Eco
Barn Pst
Eco
I
RI
HI
I
Eco
RI
RI
Results Construction of protein A / luciferaxe fusions In the first construction of plasmid pAB1, having protein A fused to the 12th amino acid of the luciferase fi subunit, a 1.1 kb EcoRI fragment from plasmid pJH5 was ligated into plasmid pRIT2T. This fusion protein did not form any enzymatically active luciferase complex when expressed together with the LYsubunit of luciferase. Fig. 1 illustrates the second construction of the hybrid protein having the protein A gene fused in-frame with the entire structural gene of the /? subunit. Plasmid pRIT2T was digested with EcoRI and treated with mung bean nuclease and the resulting plasmid pRIT2T-E was selected by its inability to be cleaved by EcoRI. 3.7 pg of each oligonucleotide (5’-GAT CCG GCT GCG ATG AAA m GGA ?TA T-K TTC CTC AAT TTA TGA ATT CTG A) and (5’-GAA TTC ATA AAA TTG AGG AAG AAT AAT CCA AAT TTC ACG CAG CCG) were mixed in a 20 ~1 reaction mixture, heated to 60” C for 5 min and then slowly cooled to room temperature to allow selfhybridization. This DNA linker, encoding the 12
t
Barn
HI
Fig. 1. Schematic representation of the construction of plasmid pAB2 coding for the protein A/B subunit fusion protein. Abbrevations: protein A (pro@& p lactamase (Ap), j3 subunit of luciferase (/WC B).
amino terminal amino acid residues of the /? subunit, was precipitated by ethanol and cloned into plasmid pRIT2T-E, previously digested with BamHI and PslI. The resulting plasmid pAABl1 was then cleaved with EcoRI and a 1.1 kb EcoRI fragment from plasmid pJH5 was inserted to give the final plasmid construction, pAB2. Furthermore, to obtain a general lux B fusion vector with multiple restriction enzyme sites in the 5’ terminal end, the Zux B gene was inserted into pUC19 as a
203
Eco RI ~ l u x A
tuxB Ap
Kan
Eco RI
J Eco RI
Pst I Hind III
Bsamm a HI Kpn I 1(
~
nll~..lClR
/ Eco RI
I Kan vu,
1
pUC19-G GAT CCG GCT GCG ATG-luxB Bam HI Fig. 2. a: Plasmid pUC19B containing the entire fl subunit of tuciferase (lux B) with multiple cloning sites in the 5' terminal end; fl lactamase (Ap). b: Construction of plasmid pJH2K coding for the a-subunit of luciferase (/ux A) and aminoglycoside 3-phosphotransferase (Kan); /~ lactamase (Ap).
P s t I - B a m H I fragment, generating p U C 1 9 B (Fig. 2a). In order to maintain b o t h the lux A and lux B plasmids in the same cell, the antibiotic resistance gene in plasmid p J H 2 had to be exchanged. Therefore, plasmid p J H 2 was digested with EcoRI and a k a n a m y c i n resistance gene was inserted into this vector. The ampicillin resistance gene in the resulting vector, p J H 2 K A , was then destroyed by deleting a 900 bp PvuI fragment. The final plasmid, p J H 2 K , thus only contained the genes encoding k a n a m y c i n resistance and the a-subunit of luciferase (Fig. 2b). Characterization of the protein A / l u c i f e r a s e conjugate The conjugate obtained from plasmids p A B 2 and p J H 2 K could be p r o d u c e d and isolated according to the protocol described in the materials and methods section. Western blotting of the conjugate showed that this was subjected to some m i n o r proteolytic degradation in vivo. However, most of the degradation products could be rem o v e d during the purification process (Fig. 3). A c c o r d i n g to S D S - P A G E , the Mr of the gene p r o d u c t s obtained from plasmid pAB1 and p A B 2
66 0 0 0 , proteinAA-subunit-
45 0 0 0
1
2
3
Fig. 3. Western blotting of the protein A/luciferase conjugate obtained from the plasmids pAB2 and pJH2K. The blotted proteins were incubated with peroxidase conjugated rabbit IgG (1/300) which is able to bind to the protein A moiety of the conjugate. This further demonstrates that the conjugate possesses IgG binding properties which are similar to native protein A. Lane /:protein extract from whole cells containing the conjugate. Lane 2: conjugate after purification on DEAESepharose. Lane 3: protein extract from whole cells containing native luciferase.
204
100
-____
90
~
80 ,-, 70
~t
Fig. 4. Schematic representation of the protein A-luciferase conjugate showing the a and fl subunit of luciferase and protein A.
2O 10 were approximately 63 k D a and 64 kDa, respectively, which agreed with our theoretical calculations. Gel filtration of the enzymatically active conjugate showed that this had no tendency to associate into multimeric aggregates. Thus, it existed only as a monomeric enzyme complex where an a subunit had been attached to each protein A / l u c i f e r a s e / 3 subunit (Fig. 4). The conjugate had an M r corresponding to 105 kDa. The specific bioluminescent activity of the conjugate was found to be approximately 1.1 )< 1014 m V / m o l , using the conditions described in the materials and methods section. A comparison with highly purified native luciferase showed that at least 50% of the specific activity was retained on the conjugate. In order to further characterize the physical properties of the protein A/luciferase complex, the conjugate was treated with 1 - 4 M urea for 60 min. As can be seen in Fig. 5, the bioluminescent activity profile in urea was essentially the same as the one obtained for the native luciferase complex. Investigations into the emitted wavelength spectrum did not show any differences between engineered and native luciferase when monitored on whole cells. The bioluminescent enzyme activity of the conjugate was stable to freezing but was observed to show reduced I g G binding capacity after storage at subzero temperatures and therefore the conjugate was routinely stored at 4 ° C , where it retained high activity for several weeks.
0
1
2
3
4
urea (mole/I) Fig. 5. Bioluminescent activity profile of native luciferase ( ) and recombinant protein A-luciferase (--) in urea. The samples were obtained from dialysed protein extract, treated with 100 m M sodium phosphate buffer p H 7.3, 1 m M D T T and urea for
60 min at 22°C.
Bioluminescent imrnunoassay In order to demonstrate the potential of the protein A/luciferase conjugate in various I g G
2O0O
.~ 1000 .Q
O~ o
,o
~o
4o
40
so
mg IgG-Sepharose
Fig. 6. Standard curve for the determination of IgG-Sepharose using the protein A-luciferase conjugate. For details see the materials and methods section.
205 binding studies, a simple bioluminescent immunoassay was set up. In these experiments different amounts of IgG-Sepharose gel were mixed with blank Sepharose gel to give a constant amount of gel in the test tube. Fig. 6 demonstrates that the fusion protein was only bound to the immobilized antibodies and that a good linearity with increasing amount of lgG-Sepharose gel was obtained within the tested range. No nonspecific binding of the conjugate to the blank Sepharose gel was observed. Furthermore, bioluminescence could be monitored down to less than 10 mV with good accuracy corresponding to a detection limit of approximately 9 x 10 -14 mol of conjugate, based on the specific activity given above.
Discussion
Luciferase has a unique potential which permits highly sensitive bioluminescent immunoassays to be developed. For instance it has previously been demonstrated that it is feasible to detect only a few hundred luciferase molecules in a sample (Hastings et al., 1978). However, the use of luciferase as a marker enzyme has hitherto found only limited application in bioluminescent immunoassays. This can be explained, in part, by the low level of bioluminescent activity obtained after the commonly used chemical crosslinking procedures, even though some milder methods have been developed (Jablonski, 1985). Therefore, the employment of gene fusion is an attractive alternative for the preparation of enzyme conjugates with a high degree of retained activity. Furthermore, these conjugates have a well-defined structure and are suitable for large scale production at a low cost. In this paper we have described the fusion between protein A and bacterial luciferase (lux B) and its application in a simple bioluminescent immunoassay. The nature of protein A indicates that the protein A/luciferase conjugate can be used as a general marker in the labelling of antibodies from many species. The preparation of a similar protein A / e n z y m e hybrid protein suggested that the IgG binding capacity of the protein A moiety had not been changed dramatically (Baneyx and Georgiou, 1989). An estimation of
the detection limit for rabbit IgG, using the protein A/luciferase conjugate, would be approximately 9 x 10 - 1 4 mol, based on a binding ratio of 1 : 1 for the protein A-IgG interaction. In order to further verify and extend the practical applicability of the protein A/luciferase conjugate we also tested it against anti-insulin antibodies. In a preliminary test system based on competitive enzyme immunoassay methodology, free insulin was able to compete with insulin immobilized on Sepharose for a limited amount of guinea pig anti-insulin antibodies present in the solution. The Sepharose mixture was centrifuged and excess conjugate was allowed to react with the antibodies that had bound to the immobilized insulin. After a final centrifugation step the insulin concentration was estimated by the bioluminescence obtained from the bound conjugate. The system was not optimized but the example given illustrates that this technique can be utilized to detect various antigens provided that the protein A moiety of the engineered conjugate has affinity to the corresponding antibody used in the assay. The observed difference in specific activity for the conjugate and native luciferase might be explained by steric hindrance around the active site of the enzyme or, alternatively, by some minor distortions in the structure of the luciferase part of the conjugate. The fusion of protein A to the entire/3 subunit did not, however, seem to change the properties of the luciferase moiety of the complex since the emitted wavelength spectrum and the sensitivity to urea were almost the same as for the native luciferase. It has previously been observed that the bioluminescence of bacterial luciferase is dependent on different factors such as the structure of the enzyme and the flavin analogue used in the reaction (Cline and Hastings, 1974). These results indicate that the general lux B fusion vector, plasmid pUC19B, could be useful in the construction of other hybrid luciferase fusion proteins. Thus, we have prepared a similar gene fusion between lux B and the galactose dehydrogenase gene (Pseudomonas fluorescens) in order to test the potential of hybrid luciferase proteins in other bioanalytical systems (Lindbladh et al., 1990). With the development of more advanced instrumentation for the simultaneous measurements of multiple samples (Maly et al., 1988) we
206
believe that the use of such recombinant luciferase conjugates in enzymatic analyses should increase in the future.
Acknowledgements This work was supported, in part, by the Swedish Board for Technical Development. Christian Johansson and Magnus Munk are gratefully acknowledged for skillful technical assistance and Dr. Jakob Donner at the molecular biology core facility, University of Lund, Sweden, for the synthesis of oligonuclotides. We also wish to thank Prof. T. Baldwin, Department of Biochemistry and Biophysics, Texas A&M University, U.S.A., for the generous gifts of plasmids pJH2 and pJH5.
References Baneyx, F. and Georgiou, G. (1989) Expression, purification and enzymatic characterization of a protein A-fl-lactamase hybrid protein. Enzyme Microbiol. Technol. 11, 559-567. Bates, D.L. (1987) Enzyme amplification in diagnostics. Tibtech 5, 204-209. Beaucage, S.L. and Caruthers, M.H. (1981) Deoxynucleoside phosphoramidites - A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 18591862. Boylan, M., Pelletier, J. and Meighen, E.A. (1989) Fused bacterial luciferase subunits catalyze light emission in eukaryotes and procaryotes. J. Biol. Chem. 264, 1915-1918. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254. Biilow, L., Ljungcrantz, P. and Mosbach, K. (1985) Preparation of a soluble bifunctional enzyme by gene fusion. Bio/Technol. 3, 821-823. Cline, T.W. and Hastings, J.W. (1974) Mutated luciferases with altered bioluminescence emission spectra. J. Biol. Chem. 249, 4668-4669. Corn, D.H., Mileham, A.J., Simson, M.I., Nealson, K.H., Rausch, S.K., Bonam, D. and Baldwin, T.O. (1985) Nucleotide sequence of the lux A gene of Vibrio harueyi and the complete amino acid sequence of the a subunit of bacterial luciferase. J. Biol. Chem. 269, 6139-6146. Gottesman, M.E., Adhya, S. and Das, A. (1980) Transcription antitermination by bacteriophage lamda N gene product. J. Mol. Biol., 140, 57-75. Guo, L.-H., Stepien, P.P., Tso, J.Y., Brousseau, R., Narang, S., Thomas, D.Y. and Wu, R. (1984) Synthesis of human
insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coil Gene 29, 251-254. Hastings, J.W., Baldwin, T.O. and Nicoli, M.Z. (1978) Bacterial luciferase: Assay, purification and properties. Methods Enzymol. 57, 135-152. Jablonski, E. (1985) The preparation of bacterial luciferase conjugates for immunoassay and application to Rubella antibody detection. Anal. Biochem. 148, 199-206. Johnston, T.C., Tompson, R.B. and Baldwin, T.O. (!986) Nucleotide sequence of the lux B gene of Vibrio harveyi and the complete amino acid sequence of the fl subunit of bacterial luciferase. J. Biol. Chem. 261, 4805-4811. Kohn, J. and Wilchek, M. (1982) A new approach (cyanotransfer) for cyanogen bromide activation of Sepharose at neutral pH, which yields activated resins, free of interfering nitrogen derivatives. Biochem. Biophys. Res. Commun. 107, 878-884. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Langone, J.J. (1982) Protein A of Staphylococcus aureus and related immunoglobuhn receptors produced by streptococci and pneumococci. Adv. Immunol. 32, 157-241. Lindbladh, C., Persson, M., Billow, L., Stahl, S. and Mosbach, K. (1987) The design of a simple competitive ELISA using human proinsulin-alkaline phosphate conjugates prepared by gene fusion. Biochem. Biophys. Res. Commun. 149, 607-614. Lindbladh, C., Persson, M., Biilow, L. and Mosbach, K. (1990) Enhanced bioluminescence obtained by a recombinant bifunctional enzyme, Galactose dehydrogenase/Bacterial luciferase. Submitted for publication. Ljungcrantz, P., Carlsson, H., MSnsson, M.O., Buckel, P., Mosbach, K. and Biilow, L. (1989) Construction of an artificial bifunctional enzyme, fl-galactosidase/galactose dehydrogenase, exhibiting efficient galactose channeling. Biochemistry 28, 8786-8792. Maly, F.E., Urwyler, a., Rolli, H.P., Dahinden, C.A. and De Weck, A.L. (1988) A single-photon imaging system for the simultaneous quantitation of luminescent emission from multiple samples. Anal. Biochem., 168, 462-469. McCapra, F. (1989) Shining a light on medical diagnostics. Chem. Br., 25, 139-141. Messing, J. (1983) New M 13 vectors for cloning. Methods Enzymol. 101, 20-78. Milby, K.H. (1985) Fluorimetric measurements in enzyme immunoassays. In: T.T. Ngo and H.M. Lenhoff (Eds.), Enzyme-Mediated Immunoassay. Plenum Press, New York, pp. 325-341. Miller, J. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Nilsson, B., Abrahmsrn, L. and Uhlrn, M. (1985) Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 4, 1075-1080. Olsson, O., Koncz, C. and Szalay, A.A. (1988) The use of the lux A gene of the bacterial luciferase operon as a reporter gene. Mol. Gen. Genet. 215, 1-9.
207 Persson, M., Bergstrand, M.G., Billow, L. and Mosbach, K. (1988) Enzyme purification by genetically attached polycycteine and polyphenylalanine affinity tails. Anal. Biochem., 172, 330-337. Smith, J.C., Derbyshire, R.B., Cook, E., Dunthorne, L., Viney, J., Brewer, S.J., Sassenfeld, H.M. and Bell, L.D. (1984) Chemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification. Gene 32, 321-327. Ugarova, N.N. and Lebedeva, O.V. (1987) Immobilized bacterial luciferase and its applications. Appl. Biochem. Biotechnol. 15, 35-51.
Waddle, J.J., Johnston, T.C. and Baldwin, T.O. (1987) Polypeptide folding and dimerization in bacterial luciferase occur by a concerted mechanism in vivo. Biochemistry 26, 4917-4921. Wienhausen, G. and DeLuca, M. (1982) Bioluminescent assays of picomole levels of various metabofites using immobilized enzymes. Anal. Biochem. 127, 380-388. Wienhausen, G., Kricka, L.J. and DeLuca M. (1987) Bioluminescent assays using coimmobilized enzymes. Methods Enzymol. 136, 82-93.
Journal of Immunological Methods, 137 (1991) 199-207 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100110X
JIM05858
Preparation of a genetically fused protein A/luciferase conjugate for use in bioluminescent immunoassays. C h r i s t e r L i n d b l a d h , K l a u s M o s b a c h a n d L e i f Biilow Department of Pure and Applied Biochemistry, Chemical Center, Universityof Lund, P.O. Box 124, S-221 O0Lund, Sweden
(Received 22 August 1990, revised received 19 October 1990, accepted 19 November 1990)
The genes encoding staphylococcal protein A and bacterial luciferase (Vibrio harveyi) were fused in-frame in order to obtain a general marker enzyme for bioluminescent immunoassays. Two constructs were made where protein A was ligated to the first and the 12th amino acid residue, respectively, of the N terminus of the fl subunit of luciferase. Only the first fusion protein encoding the entire/3 subunit was able to form an enzymatically active luciferase complex when expressed together with the a subunit. The fusion of protein A to luciferase did not notably alter the emitted wavelength spectrum or its stability to urea treatment. The fusion protein was found to retain at least 50% of the specific bioluminescent activity compared to native luciferase. In preliminary tests, this hybrid protein was shown to be useful in bioluminescent immunoassays. Key words: Protein A; Luciferase; Bioluminescence; Immunoassay; Fusion protein
Introduction
The use of radioactive- or enzyme-labelled antigens or antibodies has long been one of the best tools in the detection and quantification of biological substances. Radioimmunoassay has, however, several practical drawbacks compared to enzyme-linked immunoassays including safety, labelling and stability of the radioactive compound. This has led biochemist to further improve the
Correspondence to: C. Lindbladh, Department of Pure and Appfied Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden. Abbreviations: LB-broth, Luria-Bertani broth; DTT, dithiothreitol; IgG, immunoglobulin G; kb, ldlobase pair; Xgal, 5-bromo-4-chloro-3-indolyl-fl-D-galactopyranoside;IPTG, isopropyl-fl-D-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulphate-polyacrylarnidegel electrophoresis.
sensitivity and applicability of several enzymes which are commonly used in the labelling of conjugates. Different techniques such as the use of substrates generating fluorescent products (Milby, 1985) or coupled enzyme systems with substrate recycling (Bates, 1987) have thus been employed. Much attention has also been given to detection systems based on luminescence. These methods include the use of enzyme labels such as luciferase, alkaline phosphatase and horseradish peroxidase which together with a suitable substrate generates light (McCapra, 1989). The latter two enzymes have been utilized extensively since enzyme conjugates can easily be prepared chemically. Luciferases are also attractive marker enzymes known to be very sensitive and convenient in the detection of various enzymes and metabolites. Both bacterial and firefly luciferases have been e m p l o y e d for such b i o a n a l y t i c a l p u r p o s e s
200
(Ugarova and Lebedeva, 1987; Wienhausen et al., 1987). In order to make an immunoassay based on luciferase as a marker enzyme it is desirable to attach luciferase onto an antigen or an antibody. However, the bacterial enzyme tends to lose much of its activity when treated with the chemical crosslinking agents used in these procedures, as can be seen for instance in the immobilization of luciferase to solid supports (Wienhausen and DeLuca, 1982). The problems associated with such chemical procedures can be circumvented by preparing an in-frame gene fusion between the protein of interest and bacterial luciferase (Vibrio harveyi). In this paper we describe the construction of such a protein A/luciferase hybrid protein. The gene product obtained is a well-defined bifunctional enzyme conjugate that can be used directly in a bioluminescent immunoassay. Protein A was chosen because of its ability to bind I g G from a number of different species (Langone, 1982) and thereby create a general marker enzyme conjugate, suitable for bioluminescent immunoassays. Previously, similar artificial hybrid enzymes, prepared by gene fusions, have found wide application in protein purification (Smith et al., 1984; Nilsson et al., 1985; Persson et al., 1988), sequentially operating enzyme systems (Billow et al., 1985; Ljungcrantz et al., 1989), protein stabilization (Guo et al., 1984) and in analytical enzyme immunoassays (Lindbladh et al., 1987). Bacterial luciferase is a heterodimeric enzyme with two different subunits, a and fl encoded by the lux A and lux B genes, which initially interact as partly folded structures before the final folding takes place (Waddle et al., 1987). Both genes have been cloned and sequenced (Cohn et al., 1985; Jonston et al., 1986). The active enzyme produces light when it reacts with 02, F M N H 2 and a longchain aldehyde according to the following reaction: F M N H 2 + R C H O + 0 2 ---* F M N + R C O O H + H 2 0 + light
Due to the instability of F M N H 2, it is convenient to generate this substrate from F M N by the enzymes diaphorase or N A D ( P ) : N A D ( P ) H oxidoreductase, which use N A D H for this purpose. It is generally accepted that the single active center of luciferase is located on the a subunit. The func-
tion of the fl subunit is unknown but it is necessary for the enzymatic activity of luciferase. Recently, it has been demonstrated that it is possible to fuse at least 17 amino acid residues to the N terminus of the a subunit of luciferase. This was done in order to investigate the feasibility of using the lux A gene of bacterial luciferase as a reporter gene for gene fusions (Olsson et al., 1988). Furthermore, an enzymatically active lux A / l u x B fusion has been prepared by site-directed mutagenesis (Boylan et al., 1989). In this report we wish to describe an extension of such hybrid luciferase proteins, exemplified by the construction of a biologically active in-frame fusion between protein A and the amino terminal end of the fl subunit of luciferase and its potential use in bioluminescent immunoassays. We also describe the construction of a vector suitable for various in-frame fusions with luciferase (lux B).
Materials and methods
Enzymes and chemicals Restriction endonucleases and T 4 - D N A ligase were purchased from Boehringer Mannheim, Germany, and mung bean nuclease was from Pharmacia-LKB, Sweden. All enzymatic reactions were carried out as recommended by the manufacturer. Xgal, IPTG, N A D H , F M N and diaphorase were obtained from Boehringer Mannheim. DEAE-Sepharose and Sepharose 4B were purchased from Pharmacia-LKB and n-decyl aldehyde was from Sigma, U.S.A. The rabbit I g G antibodies used in the immunoassay were obtained from Dakopatts, Denmark. All other chemicals were of analytical grade and commercially available. Bacterial strains and plasmids E. coli JM105 (Messing, 1983) and N 4830-1 (Gottesman et al., 1980) were used as recipients for bacterial transformation of plasmids. Plasmids pUC19, p R I T 2 T and the kanamycin resistance gene were purchased from P h a r m a c i a - L K B . Plasmids pJH2 and pJH5 coding for the luciferase genes lux A and lux B, respectively, have been described previously (Waddle et al., 1987). Oligonucleotide synthesis was performed on a
201 381A D N A Synthesizer, Applied Biosystems, using the phosphoramidite method (Beaucage and Caruthers, 1981) at the molecular biology core facility, University of Lund, Sweden. The oligonucleotides were purified on a M o n o Q column. Cells were grown in an LB medium (Miller, 1972) containing ampicillin and kanamycin at 100 # g / ml and 30 ~ g / m l , respectively. Xgal, 40 /~g/ml, and IPTG, 0.1 mM, were used when necessary.
Production and purification of protein A/luciferase conjugate The bacterial culture was grown in LB medium containing ampicillin and kanamycin at 33 ° C. At ODs50 = 0.5, the culture was induced by heating to 42 o C and I P T G was added. The fermentation was stopped when ODss 0 reached 2.0. After centrifugation at 6000 x g for 5 min, the cell pellet was washed once with buffer A (100 m M phosphate buffer p H 7.3, 1 m M D T T ) and finally suspended in the same buffer containing 1 m M E D T A and 0.5 m g / m l of lysozyme. The cells were then lysed by putting the suspension on a rocking table for 30 rain at 22 ° C followed by sonication for 3 x 60 s (output 5, Sonifer B-30, Branson Sonic Power). All subsequent operations were carried out at 4 ° C. The crude protein extract was clarified by centrifugation at 12,000 x g for 5 rain, and solid ammonium sulfate was added to the supernatant until 55% saturation was reached. After centrifugation at 12,000 x g for 5 rain the protein pellet was dissolved in buffer A and dialysed overnight against the same buffer. Any precipitate was removed by centrifugation. The protein extract was then applied to a column of DEAE-Sepharose previously equilibrated with the same buffer. The fusion protein was eluted at 0.1 M NaC1 in a linear gradient of 0-0.5 M NaC1. The conjugate was further purified on a Superose 12, gel filtration column, Pharmacia-LKB, when examining its specific activity. A similar purification protocol was used for the preparation of native luciferase obtained from growing cells harboring plasmids p J H 2 and pJH5. After purification, the hybrid protein was routinely stored at 4 ° C. 1 m M D T T was added weekly.
Identification of the fusion protein S D S - P A G E was performed on a 10% polyacrylamide slab gel using a Tris-HC1 p H 8.3, dis-
continuous buffer system according to Laemmli (1970). The M r of the fusion protein was determined by comparing the relative mobility with those of the standard calibration proteins obtained from Pharmacia-LKB. Western blotting was performed as recommended by the manufacturer, JKA-Biotech, Denmark, The antibodies used in the reaction were peroxidase-conjugated rabbit immunoglobulin (Dakopats). H202 and 4-chloro1-naphthol were employed as substrates to visualize the peroxidase. The protein concentration was determined according to Bradford (1976), using bovine serum albumin as the standard.
Bioluminescence measurements Luminescent activity in mV was measured in a total volume of 200 /~1 by mixing the fusion protein with 100 m M sodium phosphate buffer at p H 7.3, 0.9% ( w / v ) NaC1, 0.1% ( w / v ) gelatin, diaphorase 0.35 U, F M N 0.03 m M and 0.001% n-decyl aldehyde, previously dissolved in ethanol. The reaction was started by the addition of N A D H to a final concentration of 0.25 m M and the peak luminescent value was measured. Luminescence from protein A/luciferase bound to IgG-Sepharose was monitored in a similar way. Bioluminescence from whole cells was monitored by adding 10/~1 n-decyl aldehyde to 0.5 ml cell suspension followed by vigorous mixing. All reactions were carried out at 22 ° C in a luminometer, Wallac 1250, or a Hitachi F-3000 fluorescence spectrophotometer.
Immobilization of lgG on Sepharose 4B The immobilization of antibodies was performed according to K o h n and Wilchek (1982), with some minor modifications. 2 g of Sepharose 4B were washed with ice cold water followed by 30% acetone and 60% acetone. The Sepharose was then suspended in 60% acetone and chilled below 0 o C. 60 mg CNBr and 850 m m o l triethylamine were added to the suspension and allowed to react for 45 s. The reaction mixture was then immediately poured into ice cold acetone, 0.1 M HC1, 50% (v/v). The mixture was subsequently transferred back to water by washing with ice cold 60% acetone followed by 30% acetone and water. The activated agarose gel was suspended in 0.1 M N a H C O 3 at p H 8.4 and 100 /~1 rabbit I g G anti-
202
bodies (Dakopats) were allowed to react overnight at 4” C. After coupling, additional reaction sites on the Sepharose gel were quenched with 0.2 M ethanolamine in 0.2 M Tris-HCl pH 8.0 for 30 min at 0°C. The IgG-Sepharose mixture was washed thoroughly with storage buffer (100 mM phosphate buffer pH 7.3, 0.9% NaCl and 0.02% NaN,) and suspended in the same buffer to give a final concentration of 200 mg IgG-Sepharose gel/ml. Bioluminescent immunoassay procedure Different amounts of IgG-Sepharose gel were mixed with pure Sepharose gel to give the same amount of gel in each test tube (50 mg). Equal amounts of purified protein A/luciferase conjugate, having an activity corresponding to 15,000 mV on the luminometer, were added to each test tube and the mixture was incubated for 3 h at 22 o C in a total volume of 350 ~1. Before monitoring the bioluminescence, the Sepharose mixture was washed twice with 2 volumes of storage buffer.
pRIT2T
I
Eco
RI
mung
bean
nucleate
Barn
Pst
HI
Eco
Barn Pst
Eco
I
RI
HI
I
Eco
RI
RI
Results Construction of protein A / luciferaxe fusions In the first construction of plasmid pAB1, having protein A fused to the 12th amino acid of the luciferase fi subunit, a 1.1 kb EcoRI fragment from plasmid pJH5 was ligated into plasmid pRIT2T. This fusion protein did not form any enzymatically active luciferase complex when expressed together with the LYsubunit of luciferase. Fig. 1 illustrates the second construction of the hybrid protein having the protein A gene fused in-frame with the entire structural gene of the /? subunit. Plasmid pRIT2T was digested with EcoRI and treated with mung bean nuclease and the resulting plasmid pRIT2T-E was selected by its inability to be cleaved by EcoRI. 3.7 pg of each oligonucleotide (5’-GAT CCG GCT GCG ATG AAA m GGA ?TA T-K TTC CTC AAT TTA TGA ATT CTG A) and (5’-GAA TTC ATA AAA TTG AGG AAG AAT AAT CCA AAT TTC ACG CAG CCG) were mixed in a 20 ~1 reaction mixture, heated to 60” C for 5 min and then slowly cooled to room temperature to allow selfhybridization. This DNA linker, encoding the 12
t
Barn
HI
Fig. 1. Schematic representation of the construction of plasmid pAB2 coding for the protein A/B subunit fusion protein. Abbrevations: protein A (pro@& p lactamase (Ap), j3 subunit of luciferase (/WC B).
amino terminal amino acid residues of the /? subunit, was precipitated by ethanol and cloned into plasmid pRIT2T-E, previously digested with BamHI and PslI. The resulting plasmid pAABl1 was then cleaved with EcoRI and a 1.1 kb EcoRI fragment from plasmid pJH5 was inserted to give the final plasmid construction, pAB2. Furthermore, to obtain a general lux B fusion vector with multiple restriction enzyme sites in the 5’ terminal end, the Zux B gene was inserted into pUC19 as a
203
Eco RI ~ l u x A
tuxB Ap
Kan
Eco RI
J Eco RI
Pst I Hind III
Bsamm a HI Kpn I 1(
~
nll~..lClR
/ Eco RI
I Kan vu,
1
pUC19-G GAT CCG GCT GCG ATG-luxB Bam HI Fig. 2. a: Plasmid pUC19B containing the entire fl subunit of tuciferase (lux B) with multiple cloning sites in the 5' terminal end; fl lactamase (Ap). b: Construction of plasmid pJH2K coding for the a-subunit of luciferase (/ux A) and aminoglycoside 3-phosphotransferase (Kan); /~ lactamase (Ap).
P s t I - B a m H I fragment, generating p U C 1 9 B (Fig. 2a). In order to maintain b o t h the lux A and lux B plasmids in the same cell, the antibiotic resistance gene in plasmid p J H 2 had to be exchanged. Therefore, plasmid p J H 2 was digested with EcoRI and a k a n a m y c i n resistance gene was inserted into this vector. The ampicillin resistance gene in the resulting vector, p J H 2 K A , was then destroyed by deleting a 900 bp PvuI fragment. The final plasmid, p J H 2 K , thus only contained the genes encoding k a n a m y c i n resistance and the a-subunit of luciferase (Fig. 2b). Characterization of the protein A / l u c i f e r a s e conjugate The conjugate obtained from plasmids p A B 2 and p J H 2 K could be p r o d u c e d and isolated according to the protocol described in the materials and methods section. Western blotting of the conjugate showed that this was subjected to some m i n o r proteolytic degradation in vivo. However, most of the degradation products could be rem o v e d during the purification process (Fig. 3). A c c o r d i n g to S D S - P A G E , the Mr of the gene p r o d u c t s obtained from plasmid pAB1 and p A B 2
66 0 0 0 , proteinAA-subunit-
45 0 0 0
1
2
3
Fig. 3. Western blotting of the protein A/luciferase conjugate obtained from the plasmids pAB2 and pJH2K. The blotted proteins were incubated with peroxidase conjugated rabbit IgG (1/300) which is able to bind to the protein A moiety of the conjugate. This further demonstrates that the conjugate possesses IgG binding properties which are similar to native protein A. Lane /:protein extract from whole cells containing the conjugate. Lane 2: conjugate after purification on DEAESepharose. Lane 3: protein extract from whole cells containing native luciferase.
204
100
-____
90
~
80 ,-, 70
~t
Fig. 4. Schematic representation of the protein A-luciferase conjugate showing the a and fl subunit of luciferase and protein A.
2O 10 were approximately 63 k D a and 64 kDa, respectively, which agreed with our theoretical calculations. Gel filtration of the enzymatically active conjugate showed that this had no tendency to associate into multimeric aggregates. Thus, it existed only as a monomeric enzyme complex where an a subunit had been attached to each protein A / l u c i f e r a s e / 3 subunit (Fig. 4). The conjugate had an M r corresponding to 105 kDa. The specific bioluminescent activity of the conjugate was found to be approximately 1.1 )< 1014 m V / m o l , using the conditions described in the materials and methods section. A comparison with highly purified native luciferase showed that at least 50% of the specific activity was retained on the conjugate. In order to further characterize the physical properties of the protein A/luciferase complex, the conjugate was treated with 1 - 4 M urea for 60 min. As can be seen in Fig. 5, the bioluminescent activity profile in urea was essentially the same as the one obtained for the native luciferase complex. Investigations into the emitted wavelength spectrum did not show any differences between engineered and native luciferase when monitored on whole cells. The bioluminescent enzyme activity of the conjugate was stable to freezing but was observed to show reduced I g G binding capacity after storage at subzero temperatures and therefore the conjugate was routinely stored at 4 ° C , where it retained high activity for several weeks.
0
1
2
3
4
urea (mole/I) Fig. 5. Bioluminescent activity profile of native luciferase ( ) and recombinant protein A-luciferase (--) in urea. The samples were obtained from dialysed protein extract, treated with 100 m M sodium phosphate buffer p H 7.3, 1 m M D T T and urea for
60 min at 22°C.
Bioluminescent imrnunoassay In order to demonstrate the potential of the protein A/luciferase conjugate in various I g G
2O0O
.~ 1000 .Q
O~ o
,o
~o
4o
40
so
mg IgG-Sepharose
Fig. 6. Standard curve for the determination of IgG-Sepharose using the protein A-luciferase conjugate. For details see the materials and methods section.
205 binding studies, a simple bioluminescent immunoassay was set up. In these experiments different amounts of IgG-Sepharose gel were mixed with blank Sepharose gel to give a constant amount of gel in the test tube. Fig. 6 demonstrates that the fusion protein was only bound to the immobilized antibodies and that a good linearity with increasing amount of lgG-Sepharose gel was obtained within the tested range. No nonspecific binding of the conjugate to the blank Sepharose gel was observed. Furthermore, bioluminescence could be monitored down to less than 10 mV with good accuracy corresponding to a detection limit of approximately 9 x 10 -14 mol of conjugate, based on the specific activity given above.
Discussion
Luciferase has a unique potential which permits highly sensitive bioluminescent immunoassays to be developed. For instance it has previously been demonstrated that it is feasible to detect only a few hundred luciferase molecules in a sample (Hastings et al., 1978). However, the use of luciferase as a marker enzyme has hitherto found only limited application in bioluminescent immunoassays. This can be explained, in part, by the low level of bioluminescent activity obtained after the commonly used chemical crosslinking procedures, even though some milder methods have been developed (Jablonski, 1985). Therefore, the employment of gene fusion is an attractive alternative for the preparation of enzyme conjugates with a high degree of retained activity. Furthermore, these conjugates have a well-defined structure and are suitable for large scale production at a low cost. In this paper we have described the fusion between protein A and bacterial luciferase (lux B) and its application in a simple bioluminescent immunoassay. The nature of protein A indicates that the protein A/luciferase conjugate can be used as a general marker in the labelling of antibodies from many species. The preparation of a similar protein A / e n z y m e hybrid protein suggested that the IgG binding capacity of the protein A moiety had not been changed dramatically (Baneyx and Georgiou, 1989). An estimation of
the detection limit for rabbit IgG, using the protein A/luciferase conjugate, would be approximately 9 x 10 - 1 4 mol, based on a binding ratio of 1 : 1 for the protein A-IgG interaction. In order to further verify and extend the practical applicability of the protein A/luciferase conjugate we also tested it against anti-insulin antibodies. In a preliminary test system based on competitive enzyme immunoassay methodology, free insulin was able to compete with insulin immobilized on Sepharose for a limited amount of guinea pig anti-insulin antibodies present in the solution. The Sepharose mixture was centrifuged and excess conjugate was allowed to react with the antibodies that had bound to the immobilized insulin. After a final centrifugation step the insulin concentration was estimated by the bioluminescence obtained from the bound conjugate. The system was not optimized but the example given illustrates that this technique can be utilized to detect various antigens provided that the protein A moiety of the engineered conjugate has affinity to the corresponding antibody used in the assay. The observed difference in specific activity for the conjugate and native luciferase might be explained by steric hindrance around the active site of the enzyme or, alternatively, by some minor distortions in the structure of the luciferase part of the conjugate. The fusion of protein A to the entire/3 subunit did not, however, seem to change the properties of the luciferase moiety of the complex since the emitted wavelength spectrum and the sensitivity to urea were almost the same as for the native luciferase. It has previously been observed that the bioluminescence of bacterial luciferase is dependent on different factors such as the structure of the enzyme and the flavin analogue used in the reaction (Cline and Hastings, 1974). These results indicate that the general lux B fusion vector, plasmid pUC19B, could be useful in the construction of other hybrid luciferase fusion proteins. Thus, we have prepared a similar gene fusion between lux B and the galactose dehydrogenase gene (Pseudomonas fluorescens) in order to test the potential of hybrid luciferase proteins in other bioanalytical systems (Lindbladh et al., 1990). With the development of more advanced instrumentation for the simultaneous measurements of multiple samples (Maly et al., 1988) we
206
believe that the use of such recombinant luciferase conjugates in enzymatic analyses should increase in the future.
Acknowledgements This work was supported, in part, by the Swedish Board for Technical Development. Christian Johansson and Magnus Munk are gratefully acknowledged for skillful technical assistance and Dr. Jakob Donner at the molecular biology core facility, University of Lund, Sweden, for the synthesis of oligonuclotides. We also wish to thank Prof. T. Baldwin, Department of Biochemistry and Biophysics, Texas A&M University, U.S.A., for the generous gifts of plasmids pJH2 and pJH5.
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207 Persson, M., Bergstrand, M.G., Billow, L. and Mosbach, K. (1988) Enzyme purification by genetically attached polycycteine and polyphenylalanine affinity tails. Anal. Biochem., 172, 330-337. Smith, J.C., Derbyshire, R.B., Cook, E., Dunthorne, L., Viney, J., Brewer, S.J., Sassenfeld, H.M. and Bell, L.D. (1984) Chemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification. Gene 32, 321-327. Ugarova, N.N. and Lebedeva, O.V. (1987) Immobilized bacterial luciferase and its applications. Appl. Biochem. Biotechnol. 15, 35-51.
Waddle, J.J., Johnston, T.C. and Baldwin, T.O. (1987) Polypeptide folding and dimerization in bacterial luciferase occur by a concerted mechanism in vivo. Biochemistry 26, 4917-4921. Wienhausen, G. and DeLuca, M. (1982) Bioluminescent assays of picomole levels of various metabofites using immobilized enzymes. Anal. Biochem. 127, 380-388. Wienhausen, G., Kricka, L.J. and DeLuca M. (1987) Bioluminescent assays using coimmobilized enzymes. Methods Enzymol. 136, 82-93.
