Expression in Escherichia coli and purification of a translocation-competent precursor of the chloroplast protein ferredoxin.
The precursor of the chloroplast protein ferredoxin from Silene pratensis was expressed in Escherichia coli. When a low copy number plasmid was used, the preferredoxin level was low, and the protein was soluble. The expression level was increased by using a high copy number plasmid. In protease-deficient cells transformed with the latter plasmid, the preferredoxin accumulated up to 1% of total protein, and it was found in insoluble aggregates. These aggregates were dissolved in 4 M urea, and the protein was purified to homogeneity. Amino-terminal sequencing confirmed the amino acid sequence as deduced from the copy DNA. However, the first methionine residue of the expected sequence was absent in E. coli. The purified precursor was readily imported by isolated chloroplasts and processed to the mature size.
Institute of Molecular Biology, University of Utrecht, The Netherlands.
M
M
Pilon
A D
AD
de Boer
S L
SL
Knols
M H
MH
Koppelman
R M
RM
van der Graaf
B
B
de Kruijff
P J
PJ
Weisbeek
D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005288:Ferredoxins / Q000235:genetics*; D017343:Genes, Plant; D007700:Kinetics; D010944:Plants / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D005288:Ferredoxins; D011498:Protein Precursors; D011994:Recombinant Proteins