Overexpression and purification of rat peroxisomal membrane protein 22, PMP22, in Pichia pastoris.
Peroxisomal membrane protein 22, PMP22, is an integral membrane protein that has four putative transmembrane-spanning regions. First reported as a major component of rat liver peroxisomal membranes and suggested to be involved in the metabolism of reactive oxygen species, its function and structure are still unknown owing to a lack of biochemical and structural experiments. Here we report the overproduction and purification of rat PMP22 (rPMP22) with the use of a methylotrophic yeast, Pichia pastoris, as a host. rPMP22 was localized not to peroxisomal membranes but to membrane compartments, such as the nuclear envelope. Highly pure rPMP22 was obtained in two steps. Several physicochemical assays indicated that the purified preparation should retain its functional structure. Furthermore, fed-batch fermentation yielded 90 mg of rPMP22 protein from 4L of culture. This is the first report to demonstrate the overproduction of a recombinant rPMP22 in the membrane compartments of P. pastoris.
Egawa
Kyoko
K
Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Shibata
Hiroyuki
H
Yamashita
Shun-ichi
S
Yurimoto
Hiroya
H
Sakai
Yasuyoshi
Y
Kato
Hiroaki
H
D000818:Animals; D019149:Bioreactors / Q000382:microbiology; D005285:Fermentation; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008830:Microbodies / Q000235:genetics / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism; D020675:Peroxisomes / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D008565:Membrane Proteins; C079231:Pxmp2 protein, rat; D011994:Recombinant Proteins