Production, Purification, and Cleavage of Tandem Repeats of

Production, Purification, and Cleavage of Tandem Repeats of Mouse
Prion Peptide 106-126
Adviser: Cheng-I Lee
Speaker: Shuei-Ling Chang
Date: 20120420
Prion diseases are fatal neurodegenerative disorders that can be infectious,
genetic, or sporadic in animals and humans. These diseases include scrapie in sheep,
bovine spongiform encephalopathy (BSE) in cattle, and sporadic and variant
Creutzfeld-Jakob disease (CJD) in humans. These diseases are due to conversion of
prion protein from the normal cellular isoform (PrPC) to pathogenic isoform (PrPSc).
PrPC is soluble, protease-sensitive, and has high α-helical content, whereas PrPSc is
insoluble, protease-resistant, and aggregated with high β-sheet content.
Research on the toxicity of PrPSc can help us to understand the mechanism of
prion diseases, and to find the therapy for these diseases. The cellular prion protein
consists of 208 amino acids (residues 23-231). A synthetic peptide 106-126 of PrP
(PrP106-126) has been used as a model to study the toxic properties of PrPSc because
this peptide is highly fibrillogenic and toxic to cultured neurons. It is believed that
PrP106-126 interacts with neuronal PrPC and subsequently converting PrPC into toxic
In this study, we used template repeated polymerase chain reaction (TR-PCR) to
develop a tandem repeats of mouse PrP106-126 interspersed by single methionine
residues that can be cleaved by cyanogen bromide (CNBr) into homogeneous peptide
units. Our preliminary results show various repeats of PrP106-126, indicating that this
TR-PCR experiment for construct the tandem repeat PrP106-126 is feasible. However,
there are some mutation and deletion in the sequence even though we used
proofreading Pfu DNA polymerase in TR-PCR experiment. We will try more
conditions to improve the accuracy of the target sequence. Then, we will subclone this
DNA fragment into an expression vector to produce fusion proteins in E. coli. The
expressed tandem repeat PrP106-126 will be purified with Ni2+-column and cleaved with
CNBr into multiple moPrP106-126 units. Subsequently, the toxic effect of recombinant
moPrP106-126 in neuron cells will be investigated. We expect the recombinant PrP106-126
that produced by E. coli can replace synthetic PrP106-126. Furthermore, this experiment
design provides a new expression system to produce short peptides.