NR ATRE
AU Gaikwad,J.U.; Sasaki,K.; Morimoto,K.; Akasaka,K.
TI Cell-free expression and characterization of mouse prion protein MoPrP(23-231)
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Structure of PrP and molecular determinants of infectivity STRCT-20
PT Konferenz-Poster
AB
Our previous work on hamster PrPc [1] showed the utility of high pressure NMR on detecting intermediate species possibly in the conversion of the cellular PrPc to the scrapie PrPSC. The cell-free expression system is expected to have an advantage of easy purification and isotope-labeling for NMR over conventional cell-based methods. Hence, we have started synthesizing the full length mouse prion protein MoPrP (23-231) by using the cell-free expression system. A linear expression construct has been generated by performing two PCRs, a gene specific first PCR for addition of overlapping regions to the sequence of interest and an overlap extension PCR for addition of T7 regulatory elements along with His6-tag. The proteins were expressed by using RTS 100 E.coli. For enhancing yield, the linear template has been converted into the circular template, expressed in RTS 500 E.coli reactions, analysed by SDS PAGE and western blotting. The proteins were purified by using anion exchange chromatography and characterized by western blotting and CD spectroscopy.
[1] Kuwata K, Li H, Yamada H, Legname G, Prusiner SB, Akasaka K, James TL, Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPsc? Biochemistry. 2002 Oct 15;41(41):12277-83.
AD Postdoc. J.U.Gaikwad, Ph.D. student K.Sasaki, Lecturer K.Morimoto, Professor K.Akasaka, Dept. of Biotech. Sci., Kinki Univ., Japan
SP englisch
PO Deutschland