title: Characterization of human colorectal cancer MDR1/P-gp Fab antibody.
abstract: In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29). Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl β-D-1-thiogalactopyranoside (IPTG). After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT) comprising residues 883-898 within the transmembrane (TM) domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied.
authors.affiliation: The Medical College of Dalian University, Dalian Economic & Technical Development Zone, Dalian 116622, China ; Department of Biochemistry and Molecular Biology, Dalian Medical University, 9 Western Section, Lvshun South Street, Lvshunkou District, Dalian 116044, China.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Departments of Medicine and Medical Microbiology, Creighton University, 601 N 30th Street, Omaha, NE 68131, USA.
authors.forename: Gary Guishan
authors.initials: GG
authors.lastname: Xiao
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, 9 Western Section, Lvshun South Street, Lvshunkou District, Dalian 116044, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Gao
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D061505:Cell Surface Display Techniques; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011994:Recombinant Proteins