Streptavidin-Aequorin Fusion Protein for Bioluminescent Immunoassay

Inouye, Satoshi; Sato, Junichi; Sasaki, Satoshi; Sahara, Yuiko

doi:10.1271/bbb.100798

Cited by 16 publications

(11 citation statements)

References 20 publications

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“…Although coreSA as an affinity tag in this CD47‐SA fusion protein could make the fusion protein stick strongly with the biotinylated agarose and separate CD47‐SA from other cell extracted crude proteins, the challenge is to elute the target fusion protein from the extremely strong affinity of biotin‐coreSA. To avoid that, the reported fusion proteins of coreSA were tagged with polyhistine and purified by immobilized metal affinity chromatography (IMAC) column …”

Section: Discussionsupporting

confidence: 94%

“…This protein also has exceptional stability against proteolysis. Due to these unique properties of coreSA, fusion proteins of coreSA have been exploited for a variety of in vitro and in vivo applications . In this study, plasmid vector pET20b(+)‐CD47‐SA (Figure B) was constructed by inserting the sequence encoding CD47ECD‐coreSA into PET20b(+) next to the pelB leader signal sequence.…”

Section: Discussionmentioning

confidence: 99%

“…Due to these unique properties of coreSA, fusion proteins of cor-eSA have been exploited for a variety of in vitro and in vivo applications. 7,8,27,28 In this study, plasmid vector pET20b(1)-CD47-SA ( Figure 1B) was constructed by inserting the sequence encoding CD47ECD-coreSA into PET20b(1) next to the pelB leader signal sequence. Competent E. coli cells transformed with this plasmid harnessed T7 expression system to express the recombinant CD47-SA fusion protein into the periplasmic space, as demonstrated by SDS-PAGE analysis of cell lysates in Figure 2.…”

Section: Discussionmentioning

confidence: 99%

“…To avoid that, the reported fusion proteins of cor-eSA were tagged with polyhistine and purified by immobilized metal affinity chromatography (IMAC) column. 7,8,27,28 Instead of using IMAC column to separate target protein from crude proteins, we investigated a variety of approaches to purify CD47-SA fusion protein using biotin-agarose affinity chromatography. As shown in Figure 3A, three typical approaches (varying pH, using biotin, applying chaotropic guanidine-HCl) were not able to purify CD47-SA fusion protein due to the remarkably strong interaction between cor-eSA and biotin.…”

Section: Discussionmentioning

confidence: 99%

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Purification of CD47‐streptavidin fusion protein from bacterial lysate using biotin–agarose affinity chromatography

Salehi

Peng

2016

Biotechnology Progress

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CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016.

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Section: Discussionsupporting

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Purification of CD47‐streptavidin fusion protein from bacterial lysate using biotin–agarose affinity chromatography

Salehi

Peng

2016

Biotechnology Progress

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“…The fusion protein streptavidin to aequorin (STA-AQ) [ 85 ] and minimum-sized core streptavidin to obelin (SAV-OL) [ 86 ] were highly purified from the inclusion bodies in Escherichia coli ( E. coli ) cells and applied to a bioluminescent sandwich immunoassay. The obtained hybrids demonstrated several advantages over similar chemical conjugates: the easiest way of preparation; maximum retention of properties of the initial protein; and as a result, the higher sensitivity of the assay.…”

Section: Analytical Application Of Ca 2+ -Regulmentioning

confidence: 99%

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

Krasitskaya

Bashmakova

Frank

2020

IJMS

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: The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate—coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization—Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme–substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.

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Facile Synthesis and Characterization of a Novel Tamavidin‐Luciferase Reporter Fusion Protein for Universal Signaling Applications

et al. 2020

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partners are equally soluble during expression. [2] Traditionally, proteins that were difficult to express as a fusion were purified independently and then fused using chemical conjugation techniques. This is often the only recourse when the necessity of production-level synthesis does not overlap with an available bacterial expression system. While chemical conjugations are relatively simple to generate using the large variety of specialized crosslinkers available, it is often much more difficult to control the conjugation stoichiometry and generate reproducible signals at low concentrations.This has been the case for probably the most ubiquitous immobilization proteins in current use, avidin-like proteins. These proteins provide an extremely high affinity and selective binding interaction with their small-molecule ligand biotin, and the biotin/avidin interaction remains one of the simplest methods for noncovalent linkage. Chemical fusions are common in these systems, with associated increases in purification and stoichiometric complexity as well as decreases in total yield proportional to the number of steps after expression. Although genetic fusions can be more difficult to develop and optimize initially, they result in completely reproducible stoichiometry for straightforward and sensitive quantitative analysis [3] while being available for use almost immediately following purification rather than requiring additional reactions and expensive cleanup steps that are often necessary for chemical conjugations. However, avidin-like proteins have been particularly difficult to express and purify in a bacterial system, and although there are limited reports of soluble fusion construct expression, [4] other reports still indicate challenges [5] and provide justification for the current standard of chemically fused, avidin-like protein conjugates.To combine the general utility of a luminescent reporter/ avidin-like fusion construct with the flexibility and high yields of bacterial expression, we report a novel universal reporter/ imaging construct composed of the recently described [6] biotinbinding protein tamavidin 2 (TA2) and the extremely small and bright luminescent reporter Gaussia luciferase (Gluc) to generate the fusion protein TA2Gluc. This genetic fusion pair was chosen based on many factors, not the least of which was the previously demonstrated bacterial expression of TA2. Further, Gluc is also one of the smallest luciferases and represents a Despite the avidin/biotin reaction being one of the most ubiquitous noncovalent immobilization and sensing strategies in scientific research, the ability to synthesize useful amounts of biotin-binding fusion constructs is hampered by poor solubility in bacterial expression systems. As such, there are few reports of successful genetic reporter fusions incorporating a biotin-binding partner. To address this, a sensitivity-enhanced, synthetically facile reporter fusion is developed to merge the bioluminescence output of Gaussia luciferase (Gluc) with the recently charac...

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Streptavidin-Aequorin Fusion Protein for Bioluminescent Immunoassay

Cited by 16 publications

References 20 publications

Purification of CD47‐streptavidin fusion protein from bacterial lysate using biotin–agarose affinity chromatography

Purification of CD47‐streptavidin fusion protein from bacterial lysate using biotin–agarose affinity chromatography

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

Facile Synthesis and Characterization of a Novel Tamavidin‐Luciferase Reporter Fusion Protein for Universal Signaling Applications

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