Role of transit peptide sequence of plastocyanin for its expression, processing, and copper-binding activity in Escherichia coli.
Plastocyanin is a copper protein that functions as an electron carrier in the thylakoid lumen of the chloroplast. To characterize the transit peptide of plastocyanin and develop expression systems for it in Escherichia coli, three kinds of expression vectors which encode different size precursor plastocyanin molecules were constructed. Their expression, processing, and copper-binding activity have been examined. When the full-length cDNA encoding the precursor plastocyanin from Silene pratensis was expressed in E. coli, a large amount of precursor plastocyanin accumulated in insoluble aggregates. Its accumulation level was increased by the addition of copper ions. About six percent of precursor plastocyanin molecules were transported into the periplasmic space and processed to the mature protein. On the other hand, expression of the intermediate size cDNA, which contains the hydrophobic domain and basic amino acid of C-terminal transit peptide, caused exclusive translocation to the periplasmic space and correct processing to the mature size. The addition of copper ions increased the holo-protein content, but did not change the polypeptide content of mature plastocyanin, indicating that translocation and processing are independent of the incorporation of copper ions. The mature plastocyanin content corresponds to 8% (w/w) of the total E. coli protein content (123 mg per liter of culture). The purified mature holo-protein showed almost the same spectroscopic and kinetic properties as those of purified spinach plastocyanin. Expression of the cDNA encoding the mature polypeptide and two preceeding amino acid residues caused the accumulation of only a small amount of plastocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)
Hibino
T
T
Department of Chemistry, Faculty of Science & Technology, Meijo University, Aichi.
Lee
B H
BH
Takabe
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D010970:Plastocyanin / Q000235:genetics* / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011498:Protein Precursors; D011994:Recombinant Proteins; D003300:Copper; D010970:Plastocyanin