title: Structure prediction, expression, and antigenicity of c-terminal of GRP78.
abstract: Glucose-regulated protein 78 (GRP78) is a typical endoplasmic reticulum luminal chaperone having a main role in the activation of the unfolded protein response. Because of hypoxia and nutrient deprivation in the tumor microenvironment, expression of GRP78 in these cells becomes higher than the native cells, which makes it a suitable candidate for cancer targeting. Suppression of survival signals by antibody production against C-terminal domain of GR78 (CGRP) can induce apoptosis of cancer cells. The aim of this study was in silico analysis, recombinant production, and characterization of CGRP in Escherichia coli. Structural prediction of CGRP by bioinformatics tools was done and the construct containing optimized sequence was transferred to E. coli T7 shuffle. Expression was induced by isopropyl-β-d-thiogalactoside, and recombinant protein was purified by Ni-NTA agarose resin. The content of secondary structures was obtained by circular dichroism (CD) spectrum. CGRP immunogenicity was evaluated from the immunized mouse sera. SDS-PAGE analysis showed CGRP expression in E. coli. CD spectrum also confirmed prediction of structures by bioinformatics tools. The enzyme-linked immunosorbent assay using sera from immunized mice revealed CGRP as a good immunogen. The results obtained in this study showed that the structure of truncated CGRP is very similar to its structure in the whole protein context. This protein can be used in cancer researches.
authors.affiliation: Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Aghamollaei
authors.affiliation: Department of Biology, Faculty of Basic Science, Shahed University, Tehran, Iran.
authors.forename: Seyed Latif
authors.initials: SL
authors.lastname: Mousavi Gargari
authors.affiliation: Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Ghanei
authors.affiliation: Medical Biotechnology Department, Faculty of Medical sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Mohamad Javad
authors.initials: MJ
authors.lastname: Rasaee
authors.affiliation: Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Jafar
authors.initials: J
authors.lastname: Amani
authors.affiliation: Department of Pharmaceutical Biotechnology, Faculty of pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Bakherad
authors.affiliation: Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Farnoosh
mesh_terms: D000818:Animals; D000091342:Endoplasmic Reticulum Chaperone BiP; D018985:Epitopes, B-Lymphocyte / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D018985:Epitopes, B-Lymphocyte; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D011994:Recombinant Proteins
keywords: GRP78; bioinformatics; cancer; prediction; recombinant