Engineering of a bioluminescent antigen-binding protein (original) (raw)
Related papers
Journal of Immunological Methods, 1991
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.
Applied and environmental microbiology, 1996
We expressed the luc gene, encoding luciferase from Photinus pyralis, in Staphylococcus aureus Cowan I downstream of the plasmid-borne promoter for protein A. Constitutive luciferase synthesis did not impair the growth rate of the host nor did it affect the stability of the plasmid. Light production started immediately after addition of luciferin. The kinetic profile is of the glowing rather than the peak type. Because S. aureus Cowan I produces large quantities of protein A, of which a substantial part becomes covalently attached to rigid cell walls, the bacterial cells could be specifically immobilized on a substrate to which immunoglobulin G molecules were adsorbed either directly or as secondary antibodies. Light production from these cells can be used as a reporter tool for the detection of antigen-antibody complexes. Fourfold amplifications of the emitted signals were obtained by in situ incubation of the bound cells in bacterial growth medium.
A streptavidin–luciferase fusion protein: comparisons and applications
Biomolecular Engineering, 1999
Luciferases are unique enzymes in being capable of emitting visible light as one of the end-products of their catalysis. Both procaryotic and eucaryotic organisms exist that emit light, and the luciferases from these organisms differ considerably in size as well as chemistry of catalysis. Two main, i.e. most studied groups, are the bacterial luciferases of e.g. Vibrio fisheri, Vibrio har6eyi, and Photorhabdus luminescens, responding to FMNH2, long-chain aldehyde and molecular oxygen and the insect luciferases of the fireflies Photinus pyralis and Luciola minengrelica or click beetle Pyrophorus plagiophthalamus, responding to ATP, luciferin and molecular oxygen. An emerging amount of 'new' luciferases from shrimps, fish, jelly fish and overall from marine origin, are finding their way to biotechnological applications. The common feature of these is their ability to produce light within the visible region of the spectrum, i.e. between 450 nm (blue) and 630 nm (red). In this short review, we discuss some of the recent advances on fusion proteins of eucaryotic luciferases and their applications. Special emphasis is placed on a streptavidin -luciferase fusion protein produced by insect cells using the baculovirus expression system.
The Smallest Isoform of Metridia longa Luciferase as a Fusion Partner for Hybrid Proteins
International Journal of Molecular Sciences
Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod Metridia longa. In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (KD = 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (KD = 87.6 ...
PLoS ONE, 2010
Background: The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multibicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. Methodology/Principal Findings: Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH 2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH 2 supplementation led to a 151fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. Conclusions/Significance: The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.
Fluorescent sensors based on bacterial fusion proteins
Methods and Applications in Fluorescence, 2014
Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.
2005
A chimeric antibody-binding green fluorescent protein (ZZGFPuv) was successfully constructed and applied as a powerful tool for immunological diagnosis. A gene encoding two repetitive sequences of Z-domain, derivative of IgG-binding B domain of staphylococcal protein A, was fused in-frame to the N-terminus of gfpuv gene. The chimeric gene was subsequently transformed and expressed in various strains of E. coli. Expression of chimeric protein in E. coli strain HB101 resulted in a protein translocation from cytoplasm to periplasmic space and cultivation medium. The chimeric ZZGFPuv could be purified using either IgG Sepharose column or immobilized metal (Cu 2+ ) affinity chromatography. The purified protein migrated in non-denaturing SDS-PAGE as two major bands. A fluorescent band was located at 36 kDa while another band at 48 kDa exhibited non-fluorescence. The fluorescent band was isolated and assessed for IgG-binding via fluorescent emission. The lowest amount of IgG that could be ...
2006
Genetic re-manipulation of chimeric antibody-binding green fluorescent proteins was successfully conducted to create versatile tools for immunological diagnosis. Four chimeric GFPs carrying one and two-consecutive sequences of the Fc-binding motif (Z-domain), derivative of IgG-binding B domain of Staphylococcal protein A (SpA), at the C-terminus were constructed. The chimeric Ab-binding GFPs possessed dual characteristics of both IgGbinding and activity of fluorescent emission. The chimeric proteins were purified to homogeneity using an IgG-Sepharose column. Additionally, a hexahistidine was fused to the N-terminal of the GFPZ and GFPZZ to allow a high protein recovery obtained from immobilized metal (Ni 2+ ) affinity chromatography (Ni-NTA), and for protein immobilization to the sensor surface. Results obtained from the Surface Plasmon Resonance (SPR) revealed a high binding affinity (KA) to immobilized human immunoglobulin up to 6.7 and 81.1 (10 7 /M) for the GFPZ and GFPZZ, respe...
Genetic re-manipulation of chimeric antibody-binding green fluorescent proteins was successfully conducted to create versatile tools for immunological diagnosis. Four chimeric GFPs carrying one and two-consecutive sequences of the Fc-binding motif (Z-domain), derivative of IgG-binding B domain of Staphylococcal protein A (SpA), at the C-terminus were constructed. The chimeric Ab-binding GFPs possessed dual characteristics of both IgG-binding and activity of fluorescent emission. The chimeric proteins were purified to homogeneity using an IgG-Sepharose column. Additionally, a hexahistidine was fused to the N-terminal of the GFPZ and GFPZZ to allow a high protein recovery obtained from immobilized metal (Ni2+) affinity chromatography (Ni-NTA), and for protein immobilization to the sensor surface. Results obtained from the Surface Plasmon Resonance (SPR) revealed a high binding affinity (KA) to immobilized human immunoglobulin up to 6.7 and 81.1 (107/M) for the GFPZ and GFPZZ, respectively. This affinity constant was raised up to 2-5 times higher when the chimeric GFPs harboring hexahistidine residues were captured on the sensor chip via metal coordination. The strong binding affinity to IgG of the chimeric GFPs was clinically applied to detect the antinuclear antibody. A strong intensity of fluorescence, higher than that of the classical fluorescein isothiocyanate (FITC) conjugated system, was significantly detected. Moreover, the proteins with double repeats of Fc-binding motif (GFPZZ and H6GFPZZ) obviously demonstrated a more intense fluorescent signal than those of the single Z domain, which corresponded to the result from SPR. All these findings support a high potential for applying such chimeric Ab-binding GFPs for clinical applications.