Dataset Information

Production of Ribosomal Protein S12/Renilla Luciferase Fusion and Development of a Bioluminescent Method for Detection of Aminoglycosides in Pork and Studying Its Recognition Mechanism.

ABSTRACT: In this study, the genes of Escherichia coli ribosomal protein S12 and renilla luciferase were linked and expressed to produce a fusion protein, and its intermolecular interactions and affinities with sevenaminoglycosides were studied. Then, the fusion protein was used as the core agent to develop a bioluminescent method on a conventional microplate for determination of the residues of thesevenaminoglycosides in pork. This method contained only one sample-loading step, and thus the assay was finished within 30 min. The limits of detection for the sevendrugs were in the range of 0.51-1.1 ng/mL, and the sensitivity for a specific drug was mainly determined by the receptordrug affinity but not related with the binding energy. After general comparison, the present method showed generally better performances than the previously reported enzyme-linked immunosorbent assays for aminoglycosides. This is the first study reporting the recognition mechanisms of Escherichia coli ribosomal protein S12 for aminoglycosides and developing a bioluminescent method for detection of aminoglycoside residues in pork samples.

SUBMITTER: Xia W

PROVIDER: S-EPMC9858597 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Production of Ribosomal Protein S12/Renilla Luciferase Fusion and Development of a Bioluminescent Method for Detection of Aminoglycosides in Pork and Studying Its Recognition Mechanism.

Xia Wanqiu W Zhang Lei L Wang Jianping J

Foods (Basel, Switzerland) 20230107 2

In this study, the genes of <i>Escherichia coli</i> ribosomal protein S12 and renilla luciferase were linked and expressed to produce a fusion protein, and its intermolecular interactions and affinities with sevenaminoglycosides were studied. Then, the fusion protein was used as the core agent to develop a bioluminescent method on a conventional microplate for determination of the residues of thesevenaminoglycosides in pork. This method contained only one sample-loading step, and thus the assay ...[more]

PMID: 36673376

Similar Datasets

Project description:BackgroundDown-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene.Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane.ResultsThe synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences.Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures.Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes.In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues.ConclusionsWe developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.

Project description:BackgroundAssays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data.ResultsWe developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20-85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically.ConclusionThis technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications.

Dataset Information

Production of Ribosomal Protein S12/Renilla Luciferase Fusion and Development of a Bioluminescent Method for Detection of Aminoglycosides in Pork and Studying Its Recognition Mechanism.

Publications

Production of Ribosomal Protein S12/Renilla Luciferase Fusion and Development of a Bioluminescent Method for Detection of Aminoglycosides in Pork and Studying Its Recognition Mechanism.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets