Establishment of cell free conversion system with biotin-labelled recombinant PrPsen expressed in E. coli.
OBJECTIVE
To report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope.


METHODS
A hamster PrP protein (HaPrP) was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrPSc preparation from scrapie strain 263K.


RESULTS
Protease-resistant bands were detected after four-day incubation.


CONCLUSIONS
The new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.
Zhang
Jin
J
School of Medicine, Xi'an Jiaotong University, China.
Gao
Jian-Mei
JM
Li
Feng
F
Han
Jun
J
Chen
Lan
L
Zhang
Bao-Yun
BY
Wang
Xiao-Fan
XF
Zhou
Wei
W
Liu
Yong
Y
Dong
Xiao-Ping
XP
D000818:Animals; D001710:Biotin; D015153:Blotting, Western; D002474:Cell-Free System; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis / Q000378:metabolism; D018620:PrPSc Proteins / Q000235:genetics* / Q000378:metabolism; D017096:Prion Diseases / Q000235:genetics / Q000473:pathology; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D013194:Staining and Labeling
D018620:PrPSc Proteins; D011994:Recombinant Proteins; D001710:Biotin; D010447:Peptide Hydrolases