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Purification and characterization of recombinant AY30 antifreeze protein from Leucosporidium sp.

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Title
Purification and characterization of recombinant AY30 antifreeze protein from Leucosporidium sp.
Other Titles
북극효모 유래 재조합 결빙방지단백질의 정제 및 특성규명
Authors
Lee, Sung Gu
Kim, Hak Jun
Park, Kyoung Sun
Lee, Jun Hyuck
Kang, Sung-Ho
Keywords
Antifreeze protein; Arctic yeast; Leucosporidium sp.; Molecular modeling; glycosylation
Issue Date
2011
Citation
Lee, Sung Gu, et al. 2011. Purification and characterization of recombinant AY30 antifreeze protein from Leucosporidium sp.. 한국생물공학회. 한국생물공학회. 2011.12.12~.
Abstract
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
URI
http://repository.kopri.re.kr/handle/201206/8472
Conference Name
한국생물공학회
Conference Place
한국생물공학회
Conference Date
2011.12.12~
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