title: Periplasmic fractionation of Escherichia coli yields recombinant plastocyanin despite the absence of a signal sequence.
abstract: Poplar plastocyanin has been expressed in E. coli from a synthetic gene cloned into the T7 expression system. Despite the absence of a signal sequence, large quantities of the recombinant protein were readily obtained by procedures typically used to isolate proteins from the bacterial periplasm. Several different fractionation methods were equally successful. The presence of plastocyanin in these fractions does not reflect wholesale leakage of intracellular proteins, since neither beta-galactosidase activity nor the bulk of Escherichia coli proteins were released by the fractionation. The identity of the overexpressed protein was unequivocally proven to be poplar plastocyanin by N-terminal amino acid sequence analysis and by spectroscopic characterization of the purified blue copper protein.
authors.affiliation: Department of Chemistry, Princeton University, New Jersey 08544-1009.
authors.forename: J A
authors.initials: JA
authors.lastname: Ybe
authors.forename: M H
authors.initials: MH
authors.lastname: Hecht
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic / Q000235:genetics; D008969:Molecular Sequence Data; D010970:Plastocyanin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017421:Sequence Analysis; D013045:Species Specificity; D013053:Spectrophotometry; D014197:Trees
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D010970:Plastocyanin