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A suite of polymerase chain reaction-based peptide tagging plasmids for epitope-targeted enzymatic functionalization of yeast proteins.

ABSTRACT: The budding yeast and model eukaryote Saccharomyces cerevisiae has been invaluable for purification and analysis of numerous evolutionarily conserved proteins and multisubunit complexes that cannot be readily reconstituted in Escherichia coli. For many studies, it is desirable to functionalize a particular protein or subunit of a complex with a ligand, fluorophore or other small molecule. Enzyme-catalysed site-specific modification of proteins bearing short peptide tags is a powerful strategy to overcome the limitations associated with traditional nonselective labelling chemistries. Towards this end, we developed a suite of template plasmids for C-terminal tagging with short peptide sequences that can be site-specifically functionalized with high efficiency and selectivity. We have also combined these sequences with the FLAG tag as a handle for purification or immunological detection of the modified protein. We demonstrate the utility of these plasmids by site-specifically labelling the 28-subunit core particle subcomplex of the 26S proteasome with the small molecule fluorophore Cy5. The full set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).

SUBMITTER: Nemec AA

PROVIDER: S-EPMC7316627 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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A suite of polymerase chain reaction-based peptide tagging plasmids for epitope-targeted enzymatic functionalization of yeast proteins.

Nemec Antonia A AA Tomko Robert J RJ

Yeast (Chichester, England) 20200501 5-6

The budding yeast and model eukaryote Saccharomyces cerevisiae has been invaluable for purification and analysis of numerous evolutionarily conserved proteins and multisubunit complexes that cannot be readily reconstituted in Escherichia coli. For many studies, it is desirable to functionalize a particular protein or subunit of a complex with a ligand, fluorophore or other small molecule. Enzyme-catalysed site-specific modification of proteins bearing short peptide tags is a powerful strategy to ...[more]

PMID: 32401365

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Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Dataset Information

A suite of polymerase chain reaction-based peptide tagging plasmids for epitope-targeted enzymatic functionalization of yeast proteins.

Publications

A suite of polymerase chain reaction-based peptide tagging plasmids for epitope-targeted enzymatic functionalization of yeast proteins.

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