RESUMO
Mammalian DSPs have been historically isolated either from native tissue sources or from transfected insect cell cultures via time-consuming and cumbersome protocols often yielding protein of variable quality and quantity. A facile and highly reproducible alternative methodology involving the heterologous expression and purification of soluble mammalian DSPs from E. coli, which yields highly active and functional protein of a uniform quality and quantity, free of spurious posttranslational modifications inherent to mammalian and insect cell expression systems, is described in this chapter.
Assuntos
Desmoplaquinas/genética , Desmoplaquinas/isolamento & purificação , Escherichia coli/genética , Expressão Gênica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Escherichia coli/metabolismo , Plasmídeos/genética , Solubilidade , Transformação BacterianaRESUMO
Desmoplakin (DP) is an obligate component of desmosomes, where it links the desmosomal cadherin/plakoglobin/plakophilin assembly to intermediate filaments. DP contains a large amino-terminal domain (DPNT) that binds to the cadherin/plakoglobin/plakophilin complex, a central coiled-coil domain that dimerizes the molecule, and a C-terminal domain (DPCT) that binds to intermediate filaments. DPNT contains a plakin domain, comprising a set of spectrin-like repeats. DPCT contains three plakin repeat domains, each formed by 4.5 repeats of a sequence motif known as a plakin repeat that bind to intermediate filaments. Here, we review purification, biochemical characterization, and structural analysis of the DPNT plakin domain and the DPCT plakin repeat domains.
