The Screening Affinity Polypeptides Target Hsp90 Recombinant Protein In Vitro

Purpose:Heat shock protein(Hsp)is a type of molecular chaperone that is produced when cells respond to different types of stress and protects homeostasis.Mammals Hsps can be divided into several subfamilies based on their molecular weight,namely Hsp90,Hsp70,Hsp60,Hsp40,Hsp27 and so on.The HSP90 family consists of HSP90 a and HSP90 b,which are encoded by two individual genes,and the amino acid sequences of the two are 80% homologous.HSP90 proteins constitute 2-3% of the total protein in normal cells.The expression of HSP90 is induced by stress stimulation,while HSP90β is constitutively expressed in cells.Hsp90α is the main molecular chaperone that regulates the homeostasis of cells under physiological and pathological conditions,and participates in the pathological process of variety diseases.HSP90α has considered as promising candidate for therapy of age-assicated diseases(such as Alzheimer’s disease,cardiovascular disease,malignant tumors,etc.)Senile plaques that are formed by the deposition of amyloidβ(Aβ)and neurofibrillary tangles(NFTs)of hyperphosporylated tau protein are the two important pathological features in the brain of patients with Alzheimer disease(AD).The accumulation of senile plaques causes dysfunction of synapses and neuron apoptosis.In the pathological process of AD,Hsp90α is induced to express and binds to the hyperphosphorylated tau protein and Aβ protein,participating in the fibers formation of tau and A.The cascade amplifies the accumulation of senile plaques and tau protein,thereby promoting the occurrence and development of AD.Hsp90αinhibitors not only prevent tau aggregation but also dissolve polymerized tau,which help the removal of Aβ.Hsp90αinhibitors rescue Aβ-induced cognitive impairment in mice,and protect against inflammatory damage mediated by tau and Aβ proteins.Therefore,Hsp90α may be a potential drug target for the treatment of AD patients.There are many kinds of chemical inhibitors against HSP90 a,some of which have been used in the second-phase clinical trials of cancer treatment,which encourage HSP90 a to be a drug target for Alzheimer disease.However,whether these chemical drugs can pass the blood-brain barrier and whether they are toxic to normal nerve cells or nerve supporting cells have not been carefully evaluated.Peptides are gaining more and more attention as targeted drugs,such as Conbercept which is a peptide drug for wet AMD.The polypeptide drug can activate or inhibit the function of the targeting protein through the specific interaction with the targeting proteins.Due to the following beneficial chararadteristics such as high affinity,high specificity and natural metabolism,we postulate a project to study the peptide drugs targeting Hsp90α protein.The phage 7 peptide library is usually used for peptide drugs.In this study,we screened the phage 7peptide-library by using Hsp90α as a bait and studied the specificity and affinity of the peptides to HSP90 a protein in vitro.The goal of our project is to find a therapeutic peptide drug candidate for Alzheimer disease by targeting HSP90 a and provide more options in manipulation of HSP90 activity.Methods:(1)Construction of recombinant plasmid pGEX-6P-1 / Hsp90α: Primers were designed based on the full-length Hsp90α c DNA sequence published by NCBI,and gene fragments were amplified by PCR.The Hsp90α gene fragment and plasmid pGEX-6P-1 were digested with Sma1 and Xho1 restrictive enzymes respectivel.The target fragment was linked with the vector by T 4 DNA ligase,and the recombinant plasmid of pGEX-6p-1-HSP90 a was transformed into E.coli DH5αand Positive clones were amplified,The recombinant plasmid were identified by DNA sequencing.,(2)Expression of recombinant GST-HSP90 a protein in vitro: Transformation of pGEX-6P-1 / Hsp90α to bacterial BL21.Upon the induction of IPTG,expression of GST-HSP90 a protein were extracted and purified and identified by anti-HSP90 antibody.The expression level were determined with Coomassie blue in SDS-PAGE and BCA protein kit.The.(3)Purification of Hsp90α protein: GST-Hsp90α protein was purified by affinity chromatography method mediated by GST-glutathione sepharose 4b beads,the ultrafilter concentrates and dialysates the protein to obtain the GST-Hsp90α protein with higher purity.(4)Screening for peptides that bind to Hsp90α: GST-Hsp90αproteins were coated in 96-well plates and incubated with the diluted peptide phage library.After washing away the unbound phages,the bound phages were incubate with and amplified with bacteria.The screening were performed for 3-4 round in this way.positive phage DNA were extracted for sequencing to release the peptid sequences.(5)Phage ELISA was used to determine the affinity of the polypeptide to Hsp90α in vitro.Results:1.The recombinant plasmid pGEX-6P-1 / Hsp90α plasmid was successfully constructed.2.getting 2.8 mg/ml of purified GST-Hsp90α proteins3.25 enriched phage clones were obtained from screening 107 phage-peptite library,Phage ELISA identified 4 phage clones with good affinity for Hsp90α.Conclusion:4peptides were identified to interact with HSP90 in vitro.Studying their regulation on HSP90 a chaperone activities will provide more alternative potential therapy for AD in future.