RESUMO
The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. GdIII -GdIII distances measured by double electron-electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-GdIII were indistinguishable from GdIII -GdIII distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.
Assuntos
Proteínas Ligantes de Maltose/análise , Ácidos Picolínicos/química , Selenoproteínas/química , Estrutura Molecular , Selenoproteínas/síntese químicaRESUMO
A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Anemia Infecciosa Equina/virologia , Imunodifusão/métodos , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Proteínas Ligantes de Maltose/análise , Proteínas do Core Viral/análise , Animais , Ensaio de Imunoadsorção Enzimática/instrumentação , Anemia Infecciosa Equina/diagnóstico , Anemia Infecciosa Equina/imunologia , Cavalos , Imunodifusão/instrumentação , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/imunologia , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/imunologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologiaRESUMO
We report the discovery of a highly reactive peptide tag for the specific cysteine conjugation of proteins. Screening of cysteine-containing peptides using ELISA-type screening yielded a 19-amino acid tag (DCPPPDDAADDAADDAADD), named DCP3 tag, which enabled the rapid and selective labeling of the tag-fused protein with a synthetic zinc complex on the surface of living cells.