Expression of PrP(C) as HIS-fusion form in a baculovirus system and conversion of expressed PrP-sen to PrP-res in a cell-free system.
Conversion of the PrP cellular form (PrP(C)) to the pathogenic form (PrP(Sc)) 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 PrP(Sc) 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 PrP(C) and PrP(Sc). 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 PrP(C) from mammalian or non-mammalian sources.
Zhang
Fu-Ping
FP
Institute of Virology, Chinese Academy of Preventive Medicine, Ying-Xin Road 100, Beijing 100052, People's Republic of China.
Zhang
Jin
J
Zhou
Wei
W
Zhang
Bao-Yun
BY
Hung
Tao
T
Dong
Xiao-Ping
XP
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002474:Cell-Free System; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D006801:Humans; D010455:Peptides / Q000138:chemical synthesis; D018621:PrPC Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018620:PrPSc Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014776:Virus Cultivation
D010455:Peptides; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011994:Recombinant Proteins