Heterologous expression of Ascaris suum cytochrome b5 precursor protein: a histidine-tagged full-length presequence is correctly processed to transport the mature protein to the periplasm of Escherichia coli.
The cytochrome b(5) of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a novel type of cytochrome b(5). It is a soluble protein that lacks the COOH-terminal membrane-anchoring domain found in erythrocyte cytochrome b(5), but possesses an NH(2)-terminal extension (presequence) of 30 amino acids that are missing from the 82-residue protein purified from the nematode tissues [Yu, Y., Yamasaki, H., Kita, K., and Takamiya, S., 1996, Arch. Biochem. Biophys. 328, 165-172]. The nematode cytochrome b(5) is, therefore, probably synthesized as a precursor protein whose presequence is cleaved to form a mature protein, but the localization of the mature protein is still unknown. To investigate the processing of the putative precursor protein, the wild-type precursor of nematode cytochrome b(5) with a complete presequence (b5wt) and its NH(2) terminus-truncated derivatives, b5Delta18 and b5Delta28, with 18 and 28 residues deleted, respectively, were expressed using pET-28a(+) vector in Escherichia coli. As expected, all transformants, tb5wt, tb5Delta18, and tb5Delta28, produced recombinant proteins with a histidine-tagged NH(2)-terminal extension. However, only the recombinant protein with the full-length presequence, produced in tb5wt, was correctly processed and transported to the periplasm, from which the majority of the induced product was purified as a mature protein chemically and functionally identical to the native protein purified from the nematode body wall. These results clearly show that the nematode histidine-tagged presequence functions as a signal peptide in E. coli.
Takamiya
Shinzaburo
S
Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan. stakamiy@med.juntendo.ac.jp
Yamasaki
Hiroshi
H
Hashimoto
Muneaki
M
Taka
Hikari
H
Murayama
Kimie
K
Tagaya
Mitsuo
M
Aoki
Takashi
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D017165:Ascaris suum / Q000378:metabolism*; D001483:Base Sequence; D002461:Cell Line, Transformed; D003579:Cytochrome Reductases / Q000378:metabolism; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013347:Subcellular Fractions
D000596:Amino Acids; D018076:DNA, Complementary; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase