Functional expression of monomeric streptavidin and fusion proteins in Escherichia coli: applications in flow cytometry and ELISA

Kroetsch, Andrew; Chin, Brandon; Nguyen, Vyncent; Gao, Jie; Park, Sheldon

doi:10.1007/s00253-018-9377-7

Cited by 11 publications

(8 citation statements)

References 51 publications

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“…Although the affinity of CS 3SL B for mSA was not measured in the present work, the reported affinity of biotin is 7.7 nM and is expected to remain high over a wide range of pH. 45 Prior to applying CUPRA-ZYME with CS 3SL B and mSA to assess the activity pH profile of NEU3, we first sought to compare the NEU3 hydrolysis kinetics of CS 3SL S1 and CS 3SL B at neutral pH. To do this, the assay was performed on a solution containing both CS 3SL S1 and CS 3SL B , along with their corresponding Uni P proxy (mSA and hCA1).…”

Section: ■ Results and Discussionmentioning

confidence: 72%

“…Because the affinity of CS 3SL S1 (and the resulting CP Lac S1 ) for hCA1 decreases substantially with decreasing pH, CUPRA-ZYME measurements are not easily performed near the optimal pH (approximately 4.8) of NEU3 or other neuraminidases using CS S1 and hCA1 as Uni P proxy . , This pH restriction was overcome using a CS B substrate (CS 3SL B ), which contains a biotin affinity tag (Figure ) and mSA as the Uni P proxy . Although the affinity of CS 3SL B for mSA was not measured in the present work, the reported affinity of biotin is 7.7 nM and is expected to remain high over a wide range of pH . Prior to applying CUPRA-ZYME with CS 3SL B and mSA to assess the activity pH profile of NEU3, we first sought to compare the NEU3 hydrolysis kinetics of CS 3SL S1 and CS 3SL B at neutral pH.…”

Section: Resultsmentioning

confidence: 99%

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CUPRA-ZYME: An Assay for Measuring Carbohydrate-Active Enzyme Activities, Pathways, and Substrate Specificities

Kitov

Kitova

et al. 2020

Anal. Chem.

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Carbohydrate-Active enZymes (CAZymes) are involved in the synthesis, degradation, and modification of carbohydrates. They play critical roles in diverse physiological and pathophysiological processes, have important industrial and biotechnological applications, are important drug targets, and represent promising biomarkers for the diagnosis of a variety of diseases. Measurements of their activities, catalytic pathway, and substrate specificities are essential to a comprehensive understanding of the biological functions of CAZymes and exploiting these enzymes for industrial and biomedical applications. For glycosyl hydrolases a variety of sensitive and quantitative spectrophotometric techniques are available. However, measuring the activity of glycosyltransferases is considerably more challenging. Here, we introduce CUPRA-ZYME, a versatile and quantitative electrospray ionization mass spectrometry (ESI-MS) assay for measuring the kinetic parameters of CAZymes, monitoring reaction pathways, and profiling substrate specificities. The method employs the recently developed competitive universal proxy receptor assay (CUPRA), implemented in a time-resolved manner. Measurements of the hydrolysis kinetics of CUPRA substrates containing ganglioside oligosaccharides by the glycosyl hydrolase human neuraminidase 3 served to validate the reliability of kinetic parameters measured by CUPRA-ZYME and highlight its use in establishing catalytic pathways. Applications to libraries of substrates demonstrate the potential of the assay for quantitative profiling of the substrate specificities glycosidases and glycosyltransferases. Finally, we show how the comparison of the reactivity of CUPRA substrates and glycan substrates present on glycoproteins, measured simultaneously, affords a unique opportunity to quantitatively study how the structure and protein environment of natural glycoconjugate substrates influences CAZyme activity.

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Section: ■ Results and Discussionmentioning

confidence: 72%

Section: Resultsmentioning

confidence: 99%

CUPRA-ZYME: An Assay for Measuring Carbohydrate-Active Enzyme Activities, Pathways, and Substrate Specificities

Kitov

Kitova

et al. 2020

Anal. Chem.

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“…Contrary to avidin, streptavidin is not a glycoprotein [ 133 , 134 ] and its pI is much lower, which prevents interactions with sugar receptors [ 135 ]. One of the most elegant strategies applied consisted in engineering monomeric avidin/streptavidin to monovalent biotin binding [ 136 ] and nanosized poly-avidins named Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS) [ 137 ]. Notably, the ANANAS technology along with the use of an antibody (cetuximab) to target a ligand demonstrated an in vivo therapeutic efficacy at a dose of doxorubicin below the one clinically used [ 138 ].…”

Section: Conjugation Strategies For Acnp Generationmentioning

confidence: 99%

An Overview of Antibody Conjugated Polymeric Nanoparticles for Breast Cancer Therapy

et al. 2020

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Nanoparticles (NPs) are promising drug delivery systems (DDS) for identifying and treating cancer. Active targeting NPs can be generated by conjugation with ligands that bind overexpressed or mutant cell surface receptors on target cells that are poorly or not even expressed on normal cells. Receptor-mediated endocytosis of the NPs occurs and the drug is released inside the cell or in the surrounding tissue due to the bystander effect. Antibodies are the most frequently used ligands to actively target tumor cells. In this context, antibody-based therapies have been extensively used in HER2+ breast cancer. However, some patients inherently display resistance and in advanced stages, almost all eventually progress. Functionalized NPs through conjugation with antibodies appear to be a promising strategy to optimize targeted therapies due to properties related to biocompatibility, suitable delivery control and efficiency of functionalization. This review is focused on the different strategies to conjugate antibodies into polymeric NPs. Recent antibody conjugation approaches applied to the improvement of breast cancer therapy are highlighted in this review.

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“…1 ) 60 , 61 . The mSA protein sequence has been previously validated to bind to biotin with high specificity and reproducibility 62 , 63 . Auxotrophic markers were used to enable the selection of positively transformed colonies without the use of antibiotic-resistance genes.…”

Section: Resultsmentioning

confidence: 99%

Targeted delivery of the probiotic Saccharomyces boulardii to the extracellular matrix enhances gut residence time and recovery in murine colitis

Heavey,

Hazelton,

Wang

et al. 2024

Nat Commun

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Probiotic and engineered microbe-based therapeutics are an emerging class of pharmaceutical agents. They represent a promising strategy for treating various chronic and inflammatory conditions by interacting with the host immune system and/or delivering therapeutic molecules. Here, we engineered a targeted probiotic yeast platform wherein Saccharomyces boulardii is designed to bind to abundant extracellular matrix proteins found within inflammatory lesions of the gastrointestinal tract through tunable antibody surface display. This approach enabled an additional 24–48 h of probiotic gut residence time compared to controls and 100-fold increased probiotic concentrations within the colon in preclinical models of ulcerative colitis in female mice. As a result, pharmacodynamic parameters including colon length, colonic cytokine expression profiles, and histological inflammation scores were robustly improved and restored back to healthy levels. Overall, these studies highlight the potential for targeted microbial therapeutics as a potential oral dosage form for the treatment of inflammatory bowel diseases.

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Functional expression of monomeric streptavidin and fusion proteins in Escherichia coli: applications in flow cytometry and ELISA

Cited by 11 publications

References 51 publications

CUPRA-ZYME: An Assay for Measuring Carbohydrate-Active Enzyme Activities, Pathways, and Substrate Specificities

CUPRA-ZYME: An Assay for Measuring Carbohydrate-Active Enzyme Activities, Pathways, and Substrate Specificities

An Overview of Antibody Conjugated Polymeric Nanoparticles for Breast Cancer Therapy

Targeted delivery of the probiotic Saccharomyces boulardii to the extracellular matrix enhances gut residence time and recovery in murine colitis

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