Purification and characterization of recombinant AY30 antifreeze protein from Leucosporidium sp.
- Purification and characterization of recombinant AY30 antifreeze protein from Leucosporidium sp.
- Other Titles
- 북극효모 유래 재조합 결빙방지단백질의 정제 및 특성규명
- Lee, Sung Gu
Kim, Hak Jun
Park, Kyoung Sun
Lee, Jun Hyuck
- Antifreeze protein; Arctic yeast; Leucosporidium sp.; Molecular modeling; glycosylation
- Issue Date
- Lee, Sung Gu, et al. 2011. Purification and characterization of recombinant AY30 antifreeze protein from Leucosporidium sp.. 한국생물공학회. 한국생물공학회. 2011.12.12~.
- We previously identified novel AY30 antifreeze protein from psychrophilic arctic yeast,
Leucosporidium sp. In this study, we developed an expression system allowing high-level
production and efficient purification of recombinant AY30 (rAY30). We have succeeded in
the cloning of AY30 gene and the gene product was efficiently expressed in Pichia
pastoris. Our expression system comprises N-terminal secretion signal sequence to
promote the secretion of rAY30. N-terminal sequencing of the secreted rAY30 revealed
that the signal sequence is immediately cleaved from the polypeptide once it has been
translocated into the culture media. A simple purification protocol for secreted rAY30
involved in three steps: anion-exchange chromatography, cold-finger, followed by size
exclusion chromatography on Superdex 200 column. 10 mg of rAY30 was purified to 98%
purity from 3 liter culture supernatant. Purified rAY30 was characterized using western
blot, periodic acid–Schiff (PAS) staining and circular dichroism method. Its molecular
mass difference between glycosylated and unglycosylated rAY30 and PAS staining showed
that rAY30 is a glycoprotein. Analysis of the antifreeze activity showed that
glycosylated rAY30 and unglycosylated rAY30 exhibit similar activity. This result
suggests that the glycan part of AY30 is not essential for antifreeze function. In
addition, circular dichroism spectra analy
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