scholarly journals A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays

2021 ◽  
Vol 9 ◽  
Author(s):  
Vadim R. Viviani ◽  
Jaqueline Rodrigues Silva ◽  
Paulo Lee Ho
Keyword(s):  
Fusion Protein ◽  
Western Blotting ◽  
Protein A ◽  
Firefly Luciferase ◽  
Western Blots ◽  
Promising Alternative ◽  
Ccd Imaging ◽  
Atp Assay ◽  
Luminescent Signal ◽  
Secondary Antibodies

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1–250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5–250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

A Comparison of an Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting for Detection of Peanut Mottle Virus and Peanut Stripe Virus1

1986 ◽  
Vol 13 (2) ◽  
pp. 64-67 ◽  
Author(s):  
J. L. Sherwood ◽  
H. A. Melouk
Keyword(s):  
Western Blotting ◽  
Immunosorbent Assay ◽  
Protein A ◽  
Mottle Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coat Proteins ◽  
Western Blots ◽  
Peanut Stripe Virus ◽  
Dry Milk ◽  
The Difference

Abstract Western blotting was used to detect infections of peanut cv. Tamnut 74 with peanut mottle virus (PMV) and/or peanut stripe virus (PStV). Leaf samples were ground in electrophoresis sample buffer and heated for 5 min at 95 C prior to electrophoresis in 12% polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose sheets at 100V for 45 min. Western blots were performed by first blocking unbound sites on the nitrocellulose with 5% non-fat dry milk in Tris-buffered saline (TBS), pH 7.4 for 30 min, followed by incubation in a 1/200 dilution of PMV and/or PStV antiserum in TBS (the latter antiserum provided by J. W. Demski, U. of GA) for 45 min. This was followed by incubation in protein-A-peroxidase (2 μg/mL in TBS) for 45 min, followed by 4-chloro-1-napthol plus hydrogen peroxide in TBS. As little as 25 ng of either purified PMV or PStV was detected. This was similar to the limits of detection fo the double sandwich enzyme linked immunosorbent assay (ELISA). Because of the difference in migration of the coat proteins of PMV and PStV, both viruses may be detected in plants infected with PMV and PStV. This assay can be performed in approximately 6 h when mini-gels are used for the initial electrophoretic seperation and does not require the antiserum to be fractionated or bound to an enzyme as is the case with ELISA.

Construction of a fusion protein between protein A and green fluorescent protein and its application to Western blotting

FEBS Letters ◽  
1996 ◽  
Vol 384 (2) ◽  
pp. 193-197 ◽  
Author(s):  
Takashi Aoki ◽  
Yasumitsu Takahashi ◽  
Katherine S. Koch ◽  
Hyam L. Leffert ◽  
Hiroyuki Watabe
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Western Blotting ◽  
Fluorescent Protein ◽  
Protein A ◽  
Green Fluorescent

scholarly journals Positive Selection of Specific Antibodies Produced against Fusion Proteins

2020 ◽  
Vol 3 (2) ◽  
pp. 37
Author(s):  
Lukas Kramberger-Kaplan ◽  
Tina Austerlitz ◽  
Holger Bohlmann
Keyword(s):  
Fusion Protein ◽  
Affinity Chromatography ◽  
Positive Selection ◽  
Fusion Proteins ◽  
Protein A ◽  
High Specificity ◽  
Western Blots ◽  
Specific Antibodies ◽  
E Coli ◽  
Selection Of

A method for the positive selection of specific antibodies for target proteins expressed as fusion proteins for the production of antiserum is presented. As proof of concept, the fusion protein FLAG::His::GFP::His::FLAG was expressed in Escherichia coli, purified, and used for the immunization of rabbits. The obtained serum was precleared via protein A affinity. A CusF::FLAG fusion protein was expressed in the periplasm of E. coli and purified. GFP without tags was also expressed in E. coli and purified via organic extraction. These proteins were then coupled to NHS-activated sepharose and used for the positive selection of Anti-GFP and Anti-FLAG antibodies. The obtained sera were tested for their specificity against different protein samples and fusion proteins in Western blots. A high specificity of the antibodies could be achieved by a single affinity chromatography step. In general, we advise to express the target protein with different tags and in different E. coli compartments for antibody production and affinity chromatography.

scholarly journals PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST IPTASE

HortScience ◽  
1993 ◽  
Vol 28 (4) ◽  
pp. 257F-257
Author(s):  
Sandra L. Barbour ◽  
John J. Frett
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fusion Protein ◽  
New England ◽  
Polyclonal Antibodies ◽  
Isopentenyl Transferase ◽  
Cytokinin Biosynthesis ◽  
Western Blots ◽  
Sds Page ◽  
England Biolabs ◽  
Secondary Antibodies

Isopentenyl transferase, encoded by the ipt gene of Agrobacterium tumefaciens T-DNA, is an enzyme active in cytokinin biosynthesis. The ipt gene was cloned into the pMAL-c2 vector (New England Biolabs, Beverly, MA) and expressed as a fusion protein. The production of this fusion protein was induced by a 2 hour exposure to IPTG. The fusion protein was then purified by a mini-aggregate procedure and visualized by SDS-PAGE. To verify that the correct protein was purified, antibodies specific to the conserved region of the fusion protein were used to probe a western blot. Secondary antibodies were biotinylated. Rabbits were immunized to raise polyclonal antibodies against iptase. Using a slot format blotting apparatus, serum was titered. These antibodies will be used to probe western blots from transgenic plants transformed with various ipt constructs.

scholarly journals Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

1991 ◽  
Vol 276 (3) ◽  
pp. 637-641 ◽  
Author(s):  
F F Craig ◽  
A C Simmonds ◽  
D Watmore ◽  
F McCapra ◽  
M R H White
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Cos Cells ◽  
Escherichia Coli Cells ◽  
Luminescent Signal ◽  
The Difference ◽  
Kinetics Of

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

scholarly journals Sequence Analysis and Expression of the Attachment and Fusion Proteins of Canine Distemper Virus Wild-Type Strain A75/17

1999 ◽  
Vol 73 (3) ◽  
pp. 2263-2269 ◽  
Author(s):  
Pascal Cherpillod ◽  
Karin Beck ◽  
Andreas Zurbriggen ◽  
Riccardo Wittek
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Translation Initiation ◽  
Fusion Proteins ◽  
Canine Distemper Virus ◽  
Protein A ◽  
Biological Properties ◽  
Canine Distemper ◽  
Wild Type ◽  
Attachment Protein

ABSTRACT The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

scholarly journals Improved CUT&RUN chromatin profiling and analysis tools

2019 ◽  
Author(s):  
Michael P. Meers ◽  
Terri Bryson ◽  
Steven Henikoff
Keyword(s):  
Fusion Protein ◽  
High Efficiency ◽  
Low Cost ◽  
Protein A ◽  
Micrococcal Nuclease ◽  
Permeabilized Cells ◽  
E Coli ◽  
Carry Over ◽  
Chromatin Profiling ◽  
Premature Release

AbstractWe previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein (1). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification; 2) a modified digestion protocol that inhibits premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

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