Department of Biochemistry and CRC for Diagnostic Technologies, La Trobe University, Bundoora, Victoria, 3083, Australia
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Abstract |
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Keywords: fluorescence/fusion protein/GFP/immunofluorescence/single-chain Fv
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Introduction |
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Fluorescent labels can provide high levels of sensitivity for a wide range of analytical procedures. Conjugation of antibodies with fluorescent labels has traditionally been accomplished by in vitro chemical conjugation of organic fluorophores (Hermanson, 1996). This procedure requires large quantities of purified protein and is often problematic in terms of optimizing the level of conjugation and loss of activity. Green fluorescent protein (GFP) originally isolated from the jellyfish Aequorea victoria, has been genetically fused to many proteins in various species to produce stable chimeras which apparently retain their original biological activity as well as retaining the fluorescent properties of native GFP (Chalfie et al., 1994
; Stearns, 1995
). GFP is now widely used as a reporter for gene expression and as a fusion tag to monitor protein localization within living cells (Misteli and Spector, 1997
). Although the main use of GFP fusions has been to study biological function in vivo, various properties of GFP such as its high stability indicate that GFP can also be used as a tool for in vitro studies. Several studies have described GFP in functional fusion complexes with protein A (Aoki et al., 1996a
), streptavidin (Oker-Blum et al., 1996) and neuron-specific enolase (Aoki et al., 1996b
). In this study, our objective was to synthesize a fusion protein in Escherichia coli consisting of EGFP, a red-shifted mutant of GFP that fluoresces 35 times more intensely than the wild-type GFP (Cormack et al., 1996
), genetically linked to an scFv to produce a naturally fluorescent antibody as a single protein.
The design of an EGFPscFv chimera posed some interesting questions regarding protein folding. GFP is a cytoplasmic protein that is known to fold correctly under the reducing conditions found in the cytoplasm of Aequorea victoria and other species in which it has been expressed. However scFvs expressed in E.coli are secreted proteins that remain largely unfolded until they reach the periplasmic space where enzymes responsible for disulphide bond formation reside. Therefore, a potential problem arises with fusing a eukaryotic cytoplasmic protein (EGFP) with a prokaryotic periplasmic protein (scFv), as both fusion partners must attain their native folding characteristics in order to retain both functionalities.
Expression of the EGFPscFv chimera in the bacterial periplasm would be the most convenient method of synthesis, since this would eliminate the need for further refolding steps to generate a fully functional fluorescent scFv. However, it has not previously been established whether EGFP, a mutant that has been codon optimized for mammalian expression (Haas et al., 1996), folds correctly and is fluorescent in the periplasmic space of E.coli. In this study we focused on expressing EGFP and EGFPscFv fusion partners in the periplasmic space of E.coli to aid in the simplicity of production.
An innately fluorescent antibody that can be produced as a single protein has a range of potential applications, e.g. for use in immunofluorescence, cellular-based fluorescence assays, fluorescence recovery after photobleaching studies or optically based biosensors. This relatively simple, rapid technique may be useful as an alternative to raising polyclonal antisera or relying on antibodies raised against polypeptide tags (e.g. hexahistidine or FLAG tags) and fluorescein-labelled antibodies for detection of scFvs in many of these techniques.
This is the first report describing a GFP fusion protein that can be expressed in the periplasm of bacterial cells, to form a functional chimera that can be used in vitro. We describe a simple strategy for producing fluorescent scFv antibodies that can potentially be applied to any scFv.
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Materials and methods |
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Primers were designed to amplify and clone EGFP (Clontech) with appropriate restriction sites into an E.coli expression vector pGC (Coia et al., 1996). The gene for an anti-HepBsAg scFv, referred to as 4C2, was cloned into pGC as described in detail previously (Coia et al., 1996). EGFP fusions were amplified by PCR using oligonucleotides designed to introduce appropriate restriction sites for cloning into pGC. EGFP was inserted at the N'-terminus of 4C2 to produce N'EGFPscFv and at the C'-terminus to yield C'scFvEGFP (Figure 1). Two additional constructs without the pelB leader sequence were produced for comparative purposes: N'EGFPscFv (cyto) and C'scFvEGFP (cyto). All the above constructs were transformed into E.coli Sure cells (Stratagene) and 1 ml cultures were grown in LB containing ampicillin (100 µg/ml) to logarithmic phase (OD600
1.0) at 25°C and induced with 1 mM isopropyl-ß-D-thiogalactoside (IPTG) for 4 h. Induced cells were centrifuged and the pellet was resuspended in 0.2 ml of PBS. Cells (10 µl) were dispensed on to a glass microscope slide and assessed for EGFP fluorescence by confocal microscopy (Leica TCS-NT) using fluorescein filters. Induced cells (50 µl) were diluted into 1 ml of isoton II electrolyte solution and the levels of fluorescence analysed by flow cytometry (FACS Calibur, Beckman Dickinson).
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Periplasmic preparations were prepared as described by Halfmann et al. (1993). Briefly, induced cultures (11000 ml) were centrifuged at 10 400 g for 10 min. The pellets were resuspended (120 ml) in 0.1 M TrisHCl (pH 8.0) containing 20% sucrose, 1 mM EDTA and a Complete protease inhibitor cocktail tablet (Boehringer Mannheim) and incubated on ice for 30 min. The samples were recentrifuged as above, the supernantant was discarded and the pellet resuspended in ice-cold distilled water (120 ml). The resuspended cells were incubated on ice for 10 min then centrifuged at 15 000 g for 15 min, the supernantant fraction (periplasmic extract) being retained and stored at 20°C. Periplasmic extracts were analysed for levels of EGFP fluorescence using a fluorescence spectrophotometer (Hitachi 650-10S). SDSPAGE was performed under reducing conditions and samples were transferred to PVDF membrane (Millipore). Immunoblots were blocked by incubation with 5% milk powderPBS and probed with anti-FLAG (2 µg/ml) (Kodak) or anti-GFP (1 µg/ml) (Clontech) followed by anti-mouse (0.5 µg/ml) or rabbit (1 µg/ml) antibodies conjugated to horseradish peroxidase (HRP) (Amersham), respectively. Immunoblots were developed with ECL reagent (Pierce). HPLC analysis was performed using a Superose 12 gel filtration column (30 ml, Pharmacia) using a flow rate of 0.5 ml/min in PBS. Elution peak times were compared with calibrated molecular weight markers.
Purification and antigen binding
N'EGFPscFv and scFv were purified under native conditions by metal ion affinity chromatography (IMAC) using Talon resin (Clontech) according to the manufacturer's instructions. Purified protein was eluted using an imidazole gradient (10100 mM) and fractions were analysed by SDSPAGE. For measurements of antigen binding, ELISA plates were coated overnight at 4°C with HepBsAg (10 µg/ml in PBS) (BiosPacific, Emeryville, CA) and blocked with 5% milk powderPBS for 2 h at room temperature. Samples were incubated with gentle agitation for 1 h at room temperature. After four washes with PBS0.05% Tween 20 and three washes with distilled water, anti-FLAG (2 µg/ml) or anti-GFP (1 µg/ml) antibodies were added and incubated. The plates were washed and detected with anti-mouse (0.5 µg/ml) or anti-rabbit IgG (1 µg/ml) conjugated to HRP and the ELISA was developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Sigma).
Transfection and immunofluorescence
The Ladw2 plasmid, which contains a 1790 bp fragment encoding the HepBsAg L protein (Gallina et al., 1994), was transiently transfected into COS-7 cells using lipofectin reagent (Gibco). Optimization of the transfection procedure was carried out according to the manufacturer's conditions. Briefly, COS-7 cells were grown in RPMI medium (Gibco) supplemented with 5% heat-inactivated fetal calf serum (FCS), 50 µg/ml streptomycin and 50 U/ml penicillin. Cells were transfected with 20 µg of pLadw2 DNA using 30 µl of lipofectin reagent in 6 ml of serum-free culture medium for 6 h at 37°C. After transfection, the medium was exchanged for complete RPMI medium including 5% FCS and incubated for 72 h. For fluorescence microscopy, cells were grown on cover-slips.
For immunofluorescence, cover-slips with adherent cells were fixed in 4% paraformaldehyde in PBS for 30 min at 4°C, washed four times in PBS and permeabilized in 0.2% Triton X-100PBS for 10 min. The cells were stained for 1 h with N'EGFPscFv, scFv or an anti-HepBsAg monoclonal antibody (5 µg/ml, ZMHB1) (Zymed). For N'EGFPscFv and EGFP the cover-slips were washed six times with PBS2% FCS0.2% Triton X-100 and mounted on glass slides for analysis. For the other samples, subsequent washing and detection steps were required. ScFv was detected with 30 min incubations of anti-FLAG (2 µg/ml) followed by FITC-conjugated anti-mouse Ig antiserum (8 µg/ml) (Sigma). The anti-HepBsAg antibody was probed with FITC-anti-mouse Ig. To test for background staining, adherent cells were incubated with EGFP alone or anti-FLAG and FITCanti-mouse Ig. The cover-slips were analysed by confocal microscopy.
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Results |
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To determine the optimum conditions for expression of a functional EGFPscFv chimera, we synthesized four plasmid constructs (Figure 1), consisting of EGFP at the N'- and C'-termini of the scFv with or without a pelB leader sequence which directs the fusion protein to the periplasmic space of E.coli (Pluckthun, 1990
). For comparative purposes we also cloned the EGFP alone and the scFv alone with the pelB sequence into the same vector system.
Fluorescence of EGFPscFv fusion proteins
To examine whether E.coli cells transformed with EGFPscFv fusion proteins could express EGFP and be fluorescent, periplasmic and cytoplasmic constructs were observed for EGFP expression using confocal microscopy. The results in Figure 2A show that the fluorescence distribution of induced cells expressing the various constructs clearly differs. Cells transformed with the cytoplasmic construct (Figure 2A
, ii) N'EGFPscFv (cyto) demonstrated a relatively uniform pattern of fluorescence throughout the cytoplasm, but with areas of higher fluorescence intensity at the polar regions. In contrast, cells transformed with the periplasmic N'EGFPscFv construct (Figure 2A
, i) showed a more punctate distribution of fluorescence. A similar pattern of fluorescence has been reported previously for a fluorescently labelled maltose binding protein that localized to the periplasm of E.coli (Barak et al., 1997
). The difference in distribution of EGFP fluorescence is dependent on the pelB signal sequence and this observation suggests that there is correct trafficking of the fusion proteins to the periplasmic space, which is a good indication that the fusion proteins are fluorescent in the periplasm. Furthermore, periplasmic extracts of N'- and C'-terminal constructs were in fact fluorescent as illustrated in Figure 3
.
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Characterization of EGFPscFv fusion proteins
We then investigated whether fluorescent fusion proteins could be harvested from the periplasm, by preparing periplasmic extracts and measuring the level of fluorescence (Figure 3). Despite a proportion of the fluorescence remaining in the cell pellet, there was a high level of measurable EGFP fluorescence present in the periplasmic fraction (Figure 3
) which was further characterized. The fluorescence level of both fusion proteins in Figure 3
was similar to that of EGFP which was also grown under similar conditions and extracted from the periplasm. When we compared the fluorescence level with a standard concentration of purified EGFP, we found the fluorescence level of 100 µg/ml of periplasmic extracts (for the samples in Figure 3
) was approximately equal to 2 µg/ml of purified EGFP, indicating that ~2% of the crude periplasmic extract was fluorescent. We also compared the fluorescence of periplasmic extracts with the fluorescence remaining in the cell pellet. Only 3050% of fluorescent protein was extracted from the periplasm using this procedure; the rest remained in the cell pellet. Therefore, a more efficient periplasmic extraction method may be required to recover all the fluorescent fusion protein. However, no attempt was made to optimize this procedure.
As proteolytic breakdown of the fusion proteins is possible, it was important to establish whether the fluorescence in the periplasmic fraction was due to the chimeric protein rather than free EGFP. Periplasmic extracts were analysed for the presence of full-length fusion proteins by Western blotting. In Figure 4A, a single band of the expected molecular weight (57 kDa) was observed for N'EGFPscFv. This was assumed to be the correctly expressed N'EGFPscFv chimera, although this 57 kDa band could correspond to dimers of scFv or EGFP formed after proteolysis. Since there was no evidence of dimerization of EGFP or scFv alone under reducing conditions (demonstrated by Western blotting in Figure 4A
), the most plausible conclusion is that this 57 kDa band is in fact the N'EGFPscFv fusion protein and subsequent purification supports this conclusion.
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In contrast, only a small amount of the C'scFvEGFP periplasmic fusion protein was detected (data not shown), indicating that this fusion was not as stable as the N'EGFPscFv fusion. In addition, there was no evidence of full-length fusion proteins for the cytoplasmic constructs. Therefore, the remainder of the experiments in this study were carried out using the full-length N'EGFPscFv.
Purification and binding to HepBsAg
N'EGFPscFv was purified with the aid of the hexahistidine tag using IMAC. A single band of the expected molecular weight was eluted as shown by SDSPAGE (Figure 4C). The yield of purified fusion protein was ~200 µg/l, which is lower than the yield we achieved for the scFv alone (~400 µg/l) under the same conditions. Reduced yields of chimeric fusion proteins are a common problem which has been reported previously (Kipriyanov et al., 1997
). A sample of the His-tag purified material was further purified using HPLC gel filtration and the monomeric peak collected. We measured the fluorescence of this fraction and compared it with known concentrations of a standard purified sample of EGFP to evaluate the specific activity. For 10 µg/ml of purified N'EGFPscFv the fluorescence level was approximately equal to 1.5 µg/ml of purified EGFP.
Having demonstrated that N'EGFPscFv can be produced as a full-length fluorescent chimera in the periplasm, we sought to analyse the ability of the fusion protein to bind to the native antigen. As illustrated in Figure 5, purified N'EGFPscFv binds to HepBsAg by ELISA. Although parent scFv and N'EGFPscFv both contain a FLAG tag, in the context of the fusion protein the anti-FLAG antibody did not recognize the FLAG epitope under native conditions. A similar effect was also encountered with the anti-His tag antibody. This may be as a result of the position of the tags (C'-terminal), a FLAG tag at the N'-terminus may be more useful for detection in ELISA. In contrast, both antibodies were useful for Western blotting under non-denaturing and denaturing conditions, as shown in Figure 4A and B
. Therefore, we were unable to compare directly the binding of the parent scFv and the N'EGFPscFv fusion to HepBsAg. Instead, we analysed the binding of N'EGFPscFv to HepBsAg using an anti-GFP antibody. EGFP alone was used as a control (Figure 5
). The binding of the parent scFv was visualized using an anti-FLAG antibody. Although no direct comparison of binding can be made, these data suggest that there is a dose-dependent increase in binding of N'EGFPscFv to HepBsAg which was similar to that observed for the parent scFv. There was no evidence of binding of EGFP alone to HepBsAg when detected with anti-GFP.
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Immunofluorescence analysis
In order to assess the ability of N'EGFPscFv to bind to cells expressing the native antigen. COS-7 cells were transiently transfected to express the HepBsAg L protein. The expressed protein contained a KDEL amino acid sequence which acts as an endoplasmic reticulum (ER) retrieval sequence and previous work has shown that the HepBsAg L protein is localized to an intermediate compartment between the ER and the Golgi (ERGIC) (Gallina et al., 1994).
Transfected cells were prepared for immunofluorescence analysis, probed with N'EGFPscFv and analysed by confocal microscopy (Figure 6a). The results clearly show epinuclear fluorescence distribution, which is consistent with binding of N'EGFPscFv to an ERGIC-located protein. Approximately 20% of the transfected cells stained positive, which is probably a function of the transfection efficiency. There was very little fluorescence in other areas of the cells and no fluorescence in untransfected cells stained with N'EGFPscFv (data not shown), indicating there was little non-specific binding of N'EGFPscFv to cellular components. The periplasmic extracts could be stored at 20°C for at least 1 month without a decrease in binding activity or fluorescence. These data indicate that crude periplasmic preparations of N'EGFPscFv can be used successfully for immunofluorescence analysis.
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Discussion |
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The main benefits of using the EGFPscFv fusion protein as opposed to conventional immunostaining methods are that this approach is rapid, purification is not essential and it is a relatively inexpensive method. In addition, this technique does not require the use of anti-tag antibodies (e.g. hexahistidine, FLAG) or antibodies conjugated with fluorescent labels to detect scFvs. These are often expensive reagents owing to the high production costs of purified antibodies and the limited shelf-life of fluorescent labels. Measuring EGFP does not require any special filter attachments; EGFP can easily be monitored using standard fluorescein microscope excitation/emission filter sets and is also suitable for excitation with the argon ion laser that is commonly used in FACS and confocal microscopy. EGFP has the added advantage of being more resistant to photobleaching than is fluorescein (Niswender et al., 1995). Although the EGFP signal does not make use of any associated amplification mechanism, we have found that using an EGFPscFv fusion for immunofluorescence produced a good signal comparable in brightness to those generated using antibodies labelled with FITC. Furthermore, direct labelling with EGFP can eliminate background due to non-specific binding of the primary and secondary antibodies to targets other than the antigen, which is often a problem in immunostaining (Wang and Hazelrigg, 1994
). However, EGFPscFv fusions are unlikely to replace conventional staining using FITC or other fluorescently labelled antibodies. Instead, they provide an alternative strategy primarily in the situation when an scFv is available as opposed to a monoclonal or polyclonal antibody. There is a growing number of useful scFvs emerging from the field of antibody engineering and phage display. Furthermore, scFvs with new specificities to rare or relatively non-immunogenic epitopes can be selected from large naive libraries, which avoids the limitations of hybridoma technology (Mackenzie and To, 1998
). This study has provided a rapid, relatively simple method of producing fluorescently labelled scFvs which can potentially be applied to any scFv. We have adapted an expression vector to be suitable for the generation of EGFPscFv fusion proteins using simple cloning procedures.
A more efficient periplasmic extraction method could be useful to recover more of the EGFPscFv fusion protein. However, the fluorescence remaining in the cell pellet may represent free EGFP in the cytoplasmic fraction which could conceivably be a product of proteolysis of the fusion proteins. Alternatively, the fluorescence may be due to insoluble or aggregated protein that is retained in inclusion bodies in the cellular fraction. Therefore, it may be advantageous to express this fusion protein in the cytoplasm of E.coli such that it will fold correctly and be stable in this reducing environment. Re-engineering the construct to optimize the stability and enhance the proper folding of periplasmic proteins (Worn and Pluckthun, 1998; Bothmann and Pluckthun, 1998
), may improve the yield of correctly folded monomeric protein and possibly increase the fluorescence. Further characterization of the activity of possible dimers and multimers of the fusion protein should also be analysed, since it is possible that an enhanced fluorescence level and higher avidity could be achieved.
In this study we have explored the use of the EGFPscFv chimera for immunofluorescence analysis; however, there are many other potential applications to which this system may be applied. EGFPscFvs with activity against a membrane antigen could be useful in cell binding assays or screening large populations of cells by FACS. A new generation of scFv intrabodies has emerged which are able to bind to their antigen intracellularly (Chen et al., 1994). This technique could be useful to study the intracellular location of GFP tagged intrabodies. Instead of using scFvs this system may also be applied to small polypeptides isolated using phage display or by other proteinprotein interations. A recent study described the use of GFP as a scaffold for display of conformationally constrained peptides as a method to screen a vast library for potentially useful binding peptides (Abedi et al., 1998
). Another application in which this reagent would be extremely useful is in fluorescence recovery after photobleaching experiments, to determine the lateral diffusion rates of surface proteins such as receptors (Brass et al. 1986
). In this approach, a bivalent antibody is not used owing to aggregation artifacts; therefore, monovalent scFvEGFP fusions would be ideal reagents, although the effects of dimerization of this molecule would have to be assessed. This work is also proof of principle for production of naturally fluorescent chimeras of GFP with other disulphide-constrained molecules.
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Notes |
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Acknowledgments |
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References |
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Received August 31, 1999; revised March 6, 2000; accepted March 29, 2000.