| Parkinson's disease(PD) is a degenerative disease,which caused tremor in rest, muscle rigidity and hypokinesia,a-synuclein,a member of synuclein family,is 140 amino acids long in human,and the gene is organized as 6 exons,5 of which are protein-coding.The a-synuclein proteins are predominately expressed in brain,and major component of protein inclusions called Lewy bodies,whose appearance due to unnormal aggregration of a-synuclein is a major hallmark of PD.Many genetic and cellular factors including a-synuclein gene mutation and lipid metabolism have been shown to affect the aggregration of a-synuclein and transition of a-synuclein morphogenetic states.It is helpful to uncover the mechanisms of the occurrence of PD and treatment of Parkinson disease.Bacterial cell surface display system composed of carrier protein,passenger protein and bacterial host acted is a novel biotechnological platform towards various protein level applications,which is derived from phage display system.This study aim to select the specific peptides binding to a-synuclein displayed on the cell surface of E.coli through paskint100 from phage display peptides library by using subtraction biopanning,and could be of great consequence and values for the better maintaining of the spatial structure of a-synuclein and further targeted drug discovery,molecular diagnostic reagents development for neurodegener- ation diseases as well for the study of its protein structure and functions.Sequence analysis and phylogenetic tree are constructed to analysis the features of a-synuclein and phylogenetic relationship among a-synuclein collected from different species.Considering relatively difficult to be cloned directly from organisms,here overlap extension PCR,a rapid and efficient method by site-directed mutagenesis,was introduced to clone the mutants A30P,E46K,A53T of a-synuclein and a-synuclein isoforms including a-synuclein 126,a-synuclein 112,a-synuclein 98.And mutants A30P,E46K,A53T and wild type of a-synuclein were displayed on the surface of E.coli strains based on the Enterohemorrhagic E. coli Intimin EaeA.Western Blot and SDS-PAGE were used to detect the a-synuclein expression level.The last,phage display peptide library is applied to screen the specific peptides binding to displayed a-synuclein.From phylogenetic tree constructed based on synuclein sequences from different species,these sequencences can be assigned toα,β,γthree distinct protein groups.α-synuclein,β-synuclein,γ-synuclein are orthological genes,whereasα,β,γ-synuclein in the same species are derived from duplicated genes,belonging to paralogical genes.Bioinformatics analysis suggested that the amino-acid sequence ofα-synuclein is divided into three regions:the N-terminal amphipathic region (1-60),the central hydrophobic NAC segment(61-95) and the C-terminal acidic part(96-140).The mutant sites of A30P,E46K and A53T of a-synuclein were all located at the amphoteric a helix of a-synuclein,and the mutant site of A30P was at the 2th exon of a-synuclein,whereas the mutant sites of E46K,A30P were located at the 3th exon of a-synuclein.Three different a-synuclein isoforms have been described as products of alternative splicing,a-synuclein 140 is the whole transcript of the SNCA gene,while a-synuclein 126 is characterized by the in-frame deletion of the sequence corresponding to exon 3,and a-synuclein 112 lacks the sequence corresponding to exon 5,located on the C-terminal half of the protein,whereas the a-synuclein 98 lacks the sequences corresponding to exon3 and exon 5.Mutants A30P,E46K and A53T of a-synuclein and a-synuclein isoforms including a-synuclein 126,a-synuclein 112,a-synuclein 98 were successfully cloned through site-directed mutagenesis based on overlap PCR and PCR lignation methods,respectively.Mutants A30P,E46K,A53T and wild type of a-synuclein were successfully displayed on the surface of E.coli strains based on the Enterohemorrhagic E.coli Intimin EaeA,and a-synuclein protein WT,A30P and E46K,A53T displayed on the surface of E coli.were detected successfully through Western Blot and SDS-PAGE.Phage display peptide library is applied to screen the specific sequences binding to displayeda-synuclein.In conclusion,a-synuclein,β-synuclein,γ-synuclein are orthological genes, whereasα,βandγ-synuclein in the same species are derived from duplicated genes, belonging to paralogical genes.And a-synuclein is divided into three regions:the N-terminal amphipathic region(1-60),the central hydrophobic NAC segment (61-95) and the C-terminal acidic part(96-140).The mutant sites of A30P,E46K and A53T of a-synuclein were all located at different sites.Syn-98,syn-112 and syn-126 are products of alternative splicing forms of whole transcript.Overlap PCR can be successfully used to clone mutants A30P,E46K,A53T and splicing forms of a-synuclein.And proteins displayed based on E.coli Intimin EaeA can be applied to screen the specific sequences directly.
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