The Construction And Expression Of N-terminal Mutant Mouse Prion Protein And The Effect Of Mutants On Prion Propagation

OBJECTIVE:Prion diseases are a group of fatal neurodegenerative diseases, which are caused by misfolded prion protein. The mechanism of these diseases is supposed to be the central nervous system damage led by the anti-proteinase K PrP80, which is converted from PrPc. However, the precise mechanism of PrPc-to-PrPSc conformation conversion has not been clear. We guessed that the amino acid residues 23-28 may be important to the conversion. Based on this hypothesis, we tried to make a series of mutant prion protein gene, and construct the recombinant plasmid to express mutant protein bacterailly. And then, we could test the conformation conversion of these mutant proteins via PMCA and Proteinase K digestion.METHODS:We developed the site-directed mutagenesis using designed primers. The primers could be divided into three kinds:changing the number of positive charge in prion N-terminal, changing the prion N-terminal sequence, and changing the proline site. After the mutant genes sequenced exactly, we ligated the genes to the vector. These recombinant plasmids were transformed to competent cells. And then we could add IPTG to the medium containing transformed bacterial to induce protein expression. After purification, we conducted the protein misfolding cyclic amplification (PMCA) experiment to observe differences between wild-type prion protein and mutant prion protein.RESULTS:We got 6 mutant prion genes, including deleting 6 amino acids mutant (Del-N6), replacing all basic amino acids with methionine mutant (Met 4-1), and 4 kinds of changing sequence mutants (Resl, Res2, Res3 and Res4). Six mutant genes were ligated to vector pET-22b, and induced by IPTG to express mutant prion proteins. The proteins were purified by Ni-NTA resin and used to develop serial protein misfolding cyclic amplification (PMCA) experiment with RNA and POPG. We contrasted the results of wild-type prion protein and mutant prion proteins and found that PrPSc conformation could be tested in both of Del-N6 mutant prion protein and Met 4-1 mutant prion protein. There is no obvious difference between wild-type and mutant prion proteins.CONCLUSION:The PMCA results indicate that bacterially expressed mutant prion protein can also conduct the PrPSc amplification, similar to wild-type prion protein. This result is contradictory to the bioassay and PMCA experiments using prion protein from eukaryotic cells. It may be the reason that the glycosyl in eukaryotic prion protein is a critical factor to prion protein conformation conversion, and this factor’s effect is related to N-terminal. We previously thought that RNA and lipids were the most important factors in prion protein conformation conversion. But this result suggest that there may be other factors influent this process, whose effect is related to prion protein N-terminal.