Expression, Isolation, and Characterization of a Chloroplast Targeting Peptide

• Published in: Protein expression and purification 1999-12, Vol.17 (3), p.345-350
• Main Authors: Wienk, Hans L.J., de Kruijff, Ben
• Format: Article
• Language: English
• Subjects: Chloroplasts - metabolism; Chromatography, High Pressure Liquid; Escherichia coli - metabolism; Ferredoxins - biosynthesis; Ferredoxins - chemistry; Ferredoxins - isolation & purification; Mass Spectrometry; Pisum sativum; Plant Proteins - biosynthesis; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Protein Precursors - biosynthesis; Protein Precursors - chemistry; Protein Precursors - isolation & purification; Protein Sorting Signals - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Sequence Analysis, Protein
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1999.1139

For the first time a method is described in which an N-terminal targeting peptide is isolated from Escherichia coli. After overexpression, purification, and cleavage of a fusion protein the protease-sensitive transit peptide from the chloroplast precursor protein preferredoxin could be isolated by HPLC. It was characterized by N-terminal amino acid sequencing and electrospray mass spectrometry. Its functionality was suggested by in vitro import competition experiments with isolated pea chloroplasts, in which the isolated peptide inhibited the import of radioactively labeled preferredoxin. Results from import competition experiments performed with a transit peptide deletion mutant suggested that the four extreme C-terminal amino acids lack information to interact with the chloroplast import machinery.