Fluorescence binding assay for a small peptide based on a GFP fusion protein (original) (raw)

Peptide Arrays for Highly Sensitive and Specific Antibody-Binding Fluorescence Assays

Bioconjugate Chemistry, 2002

We report a novel generation of peptide arrays fabricated by site-specific ligation of glyoxylyl peptides onto glass slides covered by a semicarbazide sol-gel layer. These arrays allowed the highly sensitive and specific detection of antibodies in very small blood samples from infected individuals using three model peptidic epitopes (HCV Core and NS4, EBV Capsid) in an immunofluorescence assay. Comparison with standard enzyme-linked immunosorbent assays (ELISAs) demonstrated a large gain in sensitivity and specificity. These unique properties, combined with the possibility to immobilize glycoproteins such as antibodies, offer the possibility to perform sandwich immunofluorescent assays in a highly parallel format.

Purification and Characterization of 6xHis Tagged Green Fluorescence Protein (GFP)

Greener Journal of Biochemistry and Biotechnology, 2017

The major purpose of this work was to determine if a protein i.e. Green Fluorescent Protein (GFP) could be tagged using an epitope (6xHis tag) through recombinant DNA technology with an expression vector (pET28a) then expressed in E. coli followed by isolation of the protein which was purified using purification steps that not only achieved the high level of purity as desired but was also less time consuming. This was proved by isolation of the recombinant plasmid DNA thus purified with the use of purification columns sequentially i.e. affinity chromatography, gel filtration and ion exchange chromatography, each purification step contributed to the purity attained in the protein which was then tested through SDS-PAGE electrophoresis. The molecular weight of GFP was determined by use of single bands formed by the elution collected from each purification fraction on stained gels through use of coomassie blue stain, the molecular weight was approximated using the protein marker provided (Invitrogen Benchmarker Protein Ladder) and was found to be 27KDa. The protein was further analysed using mass spectrometry for a more accurate molecular mass and for derivation of its structure. The method was found to be effective though a few problems were encountered but the aim of the experiment was successfully achieved.

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.

Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

Biotechnology Progress, 2011

Immobilized metal ion affinity chromatography (IMAC) using peptide affinity tags has become a popular tool for protein purification. An important feature dictating the use of a specific affinity tag is whether its structure influences the properties of the target protein to which it is attached. In this work we have studied the influence on protein stability of two novel peptide affinity tags, namely NT1A and HIT2, and compared their effect to the commonly used hexa-histidine tag, all attached to the C-terminus of a enhanced green fluorescent protein (eGFP). A comparison of the influence of C-or N-terminal orientation of the tags was also carried out by studying the NT1A tag attached at either terminus of the eGFP. Protein stability was studied utilising guanidine hydrochloride equilibrium unfolding procedures and CD and fluorescence spectroscopy. The novel peptide affinity tags, NT1A and HIT2, and the His 6 tag were found to not affect the stability of eGFP. Although these results are protein specific, they highlight, nevertheless, the need to employ suitable characterisation tools if the impact of a specific peptide tag on the folded status or stability of a recombinant tagged protein, purified by immobilized metal ion affinity chromatographic methods, are to be rigorously evaluated and the appropriate choice of peptide tag made.

Fluorescent Labeling of Antibody Fragments Using Split GFP

PLoS ONE, 2011

Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.

Fluorescent labelling of antibody fragments using split GFP

A gene fusion system for generating antibodies against short peptides

Gene, 1987

A novel method to obtain specific antibodies against short peptides is described, involving synthesis of the corresponding oligodeoxynucleotides followed by cloning into a new set of fusion vectors, pEZZ8 and pEZZ 18, based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus protein A. The soluble gene fusion product thus obtained, can be collected from the culture medium of Escherichia coli and rapidly recovered in a one-step procedure by IgG affinity chromatography. The system was used to express a fusion protein consisting of the two Z fragments and the C-terminal part [amino acids (aa) 57-701 of human insulin-like growth factor I (IGF-I). This 16-kDa protein was purified by afIimity chromatography on IgG Sepharose and antibodies were raised in rabbits. The fusion protein elicited peptide-specific antibodies, as measured by solid-phase radioimmuno assay and Western blotting, reactive with both synthetic C-terminal peptide and the native human IGF-I protein. The result suggests that the gene fusion system can be used for efficient antibody production against short peptides encoded by synthetic oligodeoxynucleotides.

Insights into the genetic re-engineering of chimeric antibody-binding green fluorescent proteins for immunological taggers

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.

Methods for the carboxyl-terminal fluorescent labeling of peptides using solid phase peptide synthesis

Tetrahedron Letters, 1989

Two general methods for labeling synthetic peptides with a 5-dimethylamino-1-napthalenesulfonyl idansyl) group at the C-terminal residue using solid phase peptide synthesis (SPPS) are described. Dansylated peptides are ideal substrates for fluorometric proteolytic enzyme assays. We have shown the utility of peptides labeled at their amino termini with a fluorescent 5-dimethylamino-l-napthalenesulfonyl (dansyl) moiety as substrates for proteolytic enzyme assays. The method involves reversephase HPLC separation of substrate and enzymically generated product which are detected and quantified fluorometrically. An example is the assay used to follow the rates of conversion of model glycine-extended peptides to C-terminal peptide amides by the peptidyl d-amidation enzyme.' It is also possible to assay enzymes which perform amino terminal peptide modifications or endoproteolytic cleavages using peptide substrates dansylated at the C-terminus. This communication outlines two strategies for the specific labeltng of peptides at the C-terminus using solid phase peptide synthesis

Potential of real-time measurement of GFP-fusion proteins

Journal of Biotechnology, 2004

Building on the basic design concepts of Randers-Eichhorn [Biotechnol. Bioeng. 55 (1997) 921], an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed. A general cloning vector for fusion expression proteins was constructed, allowing expression of both GFP and the target protein as a fusion. Cultivations were carried out at the 20 l scale with the signal from the sensor being relayed directly to the control system of the bioreactors. The production of GFP was then measured on-line, the signal was interfaced directly with other controlling parameters, thereby allowing the microbial process to be controlled directly based on recombinant protein expression. A positive expression correlation between on-line and off-line data was obtained. Protein accretion measured off-line was quantified using both LC-MS and plate reader assays. The potential of such a sensor for many aspects of process development is considerable and we have developed a working system which allows the optimisation of production conditions, for example, linking pH control directly to the fusion protein. Results are also presented that illustrate GFP does not alter the cultivation characteristics of the target protein when compared to the native construct. Whether GFP expressed as a fusion influences the solubility of the target protein is also discussed.

Fluorescence binding assay for a small peptide based on a GFP fusion protein (original) (raw)

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