Journal of lmmunological Methods, 143 (1991) 111-117 © 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50 ADONIS 0022175991002975

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JIM 06073

Protein A-streptokinase fusion protein for immunodetection of specific IgG antibodies K a r l - H e r m a n n Schmidt, Christian Klessen, W e r n e r K6hler and Horst Malke Institute of Microbiology and Experimental Therapy, Beutenbergstrafle 11, D-6900Jena, F.R.G. (Received 4 April 1991, accepted 10 June 1991)

The streptococcal streptokinase gene truncated at its 5' end was fused to regions of the staphylococcal protein A gene encoding the Fc-binding domains A and B. The resultant fusion gene, when expressed in the Escherichia coli lacPO system or under the speA expression/secretion signals in S. sanguis, specified a bifunctional hybrid protein, SPA-SKC, capable of Fc binding and plasminogen activation. When used in immunoassays designed to titrate antisera raised against bovine chymosin, human serum albumin and fibrinogen, the assay using SPA-SKC compared well with that using a commercial SPA-enzyme conjugate. The simple preparative method together with its efficacy and ease of use, make SPA-SKC a potentially valuable detector reagent in quantitative immunology. Key words: Gene fusion; Streptokinase; Protein A; Immunoassay

Introduction

The staphylococcal cell wall protein A (SPA) has important applications in qualitative and quantitative immunology due to its binding to the Fc regions of several immunoglobulins of different origin. In particular, the labeling of SPA with suitable enzymes or other tags has yielded a great

Correspondence to: H. Malke, Institut fiir Mikrobiologie und experimentelle Therapie, Beutenbergstr. 11, D-6900 Jena, F.R.G. Abbreviations: bp, base pair; CH, bovine chymosin; FBG, human fibrinogen; Fc, constant part of IgG; HRPO, horseradish peroxidase; HSA, human serum albumin; IgG, immunoglobulin G; OPD, o-phenylenediamine dihydrochloride; PBS, phosphate-buffered saline (10 mM, pH 7.2); PBST, PBS containing 0.01% Tween 20; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; SKC, streptokinase specified by the skc gene; SPA, staphylococcal protein A.

variety of commercial reagents for the assay of IgG by quantitating the activity of the indicator moiety (Langone, 1982). Furthermore, the availability of the cloned spa gene together with its complete nucleotide sequence (Uhl6n et al., 1983a, 1984) has permitted the in vitro construction of fusion genes encoding functional domains of SPA and target peptides which provide a second desired activity (Uhl6n et al., 1983b). As examples, SPA-/3-galactosidase and SPA-alkaline phosphatase hybrid proteins can replace chemical conjugates between an enzyme and SPA in the immunodetection of specific IgG (Nilsson et al., 1985). However, no systematic studies are available evaluating SPA fusion proteins as analytical reagents in immunology. Using gene fusion techniques, we have previously shown that streptokinase, the most important prokaryotic plasminogen activator, can be truncated at its N terminus and fused with for-

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eign proteins to form hybrid streptokinases with mixed functions (Klessen et al., 1988; Malke and Ferretti, 1991). With the simple and highly sensitive assay methods for streptokinase activity in mind, we extended these studies by constructing expression plasmids coding for fusion streptokinases carrying N-terminal SPA domains with Fcbinding capacity. The utility of the resultant hybrid proteins was assessed in experiments designed to quantitate IgG antibodies to chymosin, serum albumin and fibrinogen.

transformed according to LeBlanc and Hassell (1976).

Chemicals and antisera HSA, OPD and SPA conjugated with HRPO came from Sigma. Plasminogen-free FBG was purchased from Kabi. Commercial calf rennet (Hansen Lab., Copenhagen) was used to obtain electrophoretically pure CH by affinity chromatography on histidyl Sepharose (Amourache and Vijayalakshmi, 1984). Antisera to HSA, FBG and CH were raised in rabbits by subcutaneous injections with 500/xg of the antigens.

Materials and methods

Analysis of fusion proteins Plasmids and bacteria Escherichia coli JM101 (Yanisch-Perron et al., 1985) and Streptococcus sanguis strain Challis were used as bacterial hosts and grown in, respectively, LB medium (Lennox, 1955) and Todd-Hewitt broth (Difco) at 37°C. Plasmid pKM1 carried, under the lactose promoter-operator-/3-galactosidase system (lacPOZ'), a truncated streptokinase gene, 'skc, lacking codons 1 through 39 of wild-type skc but retaining the remainder of the skc coding sequence together with the transcription terminator (Klessen et al., 1988). The open reading frame vectors pKM2 and pKM3 (Klessen et al., 1988) carrying 'skc under lacPOZ' and under the erythrogenic toxin type A promoter (spePA'), respectively, were used as starting plasmids for the construction of the spa'skc fusion genes. The source of the spa gene was pRIT3 (Nilsson et al., 1985) kindly provided by Martin Lindberg. The erythromycin resistance plasmid pSM6 (Laplace et al., 1988) was used for the construction of the shuttle vector between E. coli and S. sanguis.

Recombinant DNA techniques Plasmid DNA was isolated by CsCl-ethidium bromide density gradient centrifugation according to Sambrook et al. (1989). Restriction enzymes, T4 DNA ligase and the Klenow fragment of DNA polymerase I were used as recommended by the supplier (Boehringer). Transformation of E. coli was carried out by the CaCI 2 method as described by Sambrook et al. (1989), and competent S. sanguis cells were made and

Whole cell extracts of E. coli obtained by sonication (Malke and Ferretti, 1984) or cell-free culture supernatant fluids from streptococcal cultures were subjected to SDS-PAGE on 10% gels made according to Laemmli (1970). The separated proteins were transferred to nitrocellulose membranes by electroblotting using the method of Kyhse-Andersen (1984). After blocking the membranes with non-fat dry milk (Johnson et al., 1984), they were treated with HRPO-labeled IgG to detect SPA-reactive bands. Parallel gels were treated with 2% Triton X-100, washed with water, and overlayed with casein-plasminogen agarose (Malke and Ferretti, 1984) to visualize bands with SKC activity.

Preparation of partially purified fusion proteins The cell sediments from 100 ml E. coli JM101 (pKM36) cultures were washed with saline and resuspended in 10 ml of the same solvent. After sonication, the cell extracts were cleared by centrifugation and protein was precipitated with ammonium sulfate at 70% saturation for 18 h at 4° C. After extensive dialysis against buffer containing 50 mM Tris-HC1 pH 7.4, 150 mM NaC1, 2 mM benzamidine and 1 mM cysteine, the proteins were fractionated on a Sephacryl S-200 column (2 x 90 cm). Fractions showing both Skc activity on casein-plasminogen agarose plates and binding of HPRO-labeled IgG in a dot blot assay were pooled, and the proteins precipitated with ammonium sulfate at 70% saturation. The precipitates were dissolved in 5 ml PBS and stored at 4° C until required.

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Immunoassays Using a reaction volume of 100/zl throughout, the wells of two parallel microtiter plates (MEW Polyplast, Halberstadt) were coated with the antigens (CH, FBG and HSA) at 10 /xg/ml in PBS for 2 h at 37 ° C. After washing with PBS, the wells were blocked with 1% nonfat reconstituted skim milk in PBST and washed again with PBST. Starting from an initial dilution of 1/100, serial 1 in 4 dilutions of rabbit immune sera or preimmune sera (controls) in PBST were added to the wells in quadruplicate, and the plates were incu-

bated for 2 h at 37 ° C. After washing with PBST, the wells of one plate received aliquots from a 1/100 dilution of the SPA-SKC fusion protein solution, whereas HRPO-labeled SPA was added (from a 1/500 dilution) to the second plate. All plates were incubated for 1 h at 37 °C, washed with PBST, and the SPA-SKC plates were reacted with 1% reconstituted skim milk in PBS containing variable concentrations of plasminogen. Caseinolysis was measured after 2 h of incubation at 450 nm using a microtiter plate absorbance reader (Sumal, Zeiss). The H R P O -

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6n Fig. 1. Schematic representation of plasmids and enzymes used to construct SPA-SKC expression vectors. Arrows on the plasmid maps indicate the orientation of the ampicillin (Ap) and erythromycin (Em) resistance genes used for selection. The nucleotide sequences of the tripartite fusion genes in pKM36 and pKM37 are shown in detail, with the codon n u m b e r s corresponding to those originally published (Uhl~n et al., 1983a; Malke et al., 1985; Weeks and Ferretti, 1986) and, in the case of spa and skc, being related to the mature forms of the respective proteins. The angled arrow indicates the S P E A signal peptide cleavage site. Lower case codons originate from the polylinkers (shadowed segments in pKM2 and pKM3) of the p U C plasmids used in the constructions; bold face codons are related to spa and skc. Plasmids are presented linearly and, for the sake of clarity, not drawn to scale. Genetic symbols are explained in the text.

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labeled SPA plates were developed using OPD for 30 min and absorbance measured at 495 nm. The antibody titers of the immune sera were estimated using the equation of Caufield and Schaffer (1984).

Results

Construction of expression plasmids for SPA-SKC fusion proteins 12.

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Protein A-streptokinase fusion protein for immunodetection of specific IgG antibodies.

The streptococcal streptokinase gene truncated at its 5' end was fused to regions of the staphylococcal protein A gene encoding the Fc-binding domains...
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