RESUMO
Peptide mapping was used for the quality control of different batches of the recombinant HIV proteins p24 core and p24-gp41, expressed in Escherichia coli. These proteins comprise gag and env region polypeptides of the virus and may serve as suitable components in the diagnosis of HIV infections. The proteins were digested with trypsin and the mixtures were subjected to peptide mapping to prove batch equivalence of p24-gp41 and to isolate fragments of the p24-gp41 digest that differ from those of the p24 core digest. The proteins were reduced with dithiothreitol and the cysteine residues were derivatized by addition of 4-vinylpyridine. Peptide mapping was performed by means of reversed-phase high-performance liquid chromatography. Batch equivalence was proved by comparison of the maps. Peaks present in one map but not in the other were considered to be due to sequence differences or variability in digestion.
Assuntos
Escherichia coli/genética , HIV/análise , Peptídeos/química , Sequência de Aminoácidos , Aminoácidos/análise , Cisteína/análise , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Produtos do Gene gag/química , Produtos do Gene gag/genética , HIV/genética , Proteína do Núcleo p24 do HIV , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Hidrólise , Indicadores e Reagentes , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Peptídeos/genética , Controle de Qualidade , Espectrofotometria Ultravioleta , Tripsina , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
The primary structure of calf thymus glutaredoxin was determined by analysis of the [14C]carboxymethylated protein and the proteolytic fragments obtained by treatments with trypsin, chymotrypsin, CNBr and staphylococcal Glu-specific extracellular protease. The active center has the structure Cys-Pro-Tyr-Cys, with the redox-active cysteines/half-cystines located at positions 22 and 25 in the polypeptide chain. This active center is identical in amino acid sequence and similar in position to that of Escherichia coli glutaredoxin, suggesting this structure to be typical for glutaredoxins and distinguishing them from the distantly related thioredoxins. However, the two glutaredoxins also exhibit considerable differences. Calf thymus glutaredoxin is extended at both ends and has 31% overall residue identities with the corresponding E. coli protein. In contrast to the bacterial glutaredoxin, the calf thymus protein contains two additional half-cystines/cysteine residues at positions 74 and 78, which may be of regulatory significance.