title: Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.
abstract: Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.
authors.affiliation: Laboratory of Biopharmaceutical, College of Life Science and Biopharmaceutical, Guangdong Pharmaceutical University, Guangzhou Higher Education Mega Center, Guangzhou, Guangdong 510006, PR China.
authors.forename: Meizhi
authors.initials: M
authors.lastname: Wu
authors.forename: Lin
authors.initials: L
authors.lastname: Zhao
authors.forename: Lei
authors.initials: L
authors.lastname: Zhu
authors.forename: Zhange
authors.initials: Z
authors.lastname: Chen
authors.forename: Huangjin
authors.initials: H
authors.lastname: Li
mesh_terms: D000818:Animals; D018985:Epitopes, B-Lymphocyte / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D018984:Epitopes, T-Lymphocyte / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D008297:Male; D008459:Measles virus / Q000737:chemistry / Q000235:genetics; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014760:Viral Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D018985:Epitopes, B-Lymphocyte; D018984:Epitopes, T-Lymphocyte; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors