NR AMZM
AU Zhang,F.P.; Zhang,J.; Zhou,W.; Zhang,B.Y.; Hung,T.; Dong,X.P.
TI Expression of PrPc as HIS-fusion form in a baculovirus system and conversion of expressed PrP-sen to PrPres in a cell-free system
QU Virus Research 2002 Aug; 87(2): 145-53
PT journal article
AB Conversion of the PrP cellular form (PrPc) to the pathogenic form (PrPsc) is the key step in the pathogenesis of transmissible spongiform encephalophathies. Although the mechanism of conformational conversion of PrP proteins remains uncertain, the cell-free conversion reaction and other in vitro PrP amplification tests allow it to be studied under the much quicker and simpler conditions than those of transmission bioassay in vivo. Using baculovirus expression system, wild-type hamster (HaPrP) and human PrP (HuPrP), as well as D178N mutated human PrP (HuPrPm178) were expressed in HIS-fusion form. After 35S-methionine labeling and purification with Ni-NTA agarose affinity chromatography, individual expressed PrP proteins were mixed with PrPsc isolated from hamster brain tissue infected with scrapie 263K. Protease-resistant isoform was detected in the homologous HaPrP reaction, but not in the two heterologous HuPrP preparations, implying a species-specific molecular recognition between PrPc and PrPsc. HIS-tag in HIS-HaPrP seems to have little effect on the formation of protease-resistant protein in this preparation. This system proposes a simple and protein productive-enriched way for cell-free conversion of prion proteins, as the replacement of native or genetic engineering expressed sole PrPc from mammalian or non-mammalian sources.
MH Animal; Baculoviridae/genetics; Cell-Free System; Cloning, Molecular; Conserved Sequence; Hamsters; Human; Peptides/chemical synthesis; PrPc Proteins/chemistry/genetics/*metabolism; PrPsc Proteins/chemistry/*metabolism; Protein Conformation; Recombinant Proteins/genetics/metabolism; Spodoptera; Support, Non-U.S. Gov't; Virus Cultivation
AD Institute of Virology, Chinese Academy of Preventive Medicine, Ying-Xin Road 100, Beijing 100052, People's Republic of China.
SP englisch
PO Niederlande