Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines.
We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism.
Okumura
Fumihiko
F
Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan.
Kameda
Hiroyuki
H
Ojima
Takao
T
Hatakeyama
Shigetsugu
S
D000818:Animals; D002460:Cell Line; D002480:Cellulase / Q000096:biosynthesis* / Q000302:isolation & purification; D003470:Culture Media / Q000737:chemistry; D006801:Humans; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D047269:Strongylocentrotus / Q000201:enzymology*
D003470:Culture Media; D011994:Recombinant Proteins; D002480:Cellulase