title: Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris.
abstract: The methylotrophic yeast Pichia pastoris has become a highly popular expression host system for the recombinant production of a wide variety of proteins, such as antibody fragments. Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy chain antibody (VHH). To test the feasibility of expressing VHHs in the yeast, which on account of their small size and antigen recognition properties would have a major impact on antibody engineering strategies, we constructed two VHH genes encoding the single-domain antibody fragments with specificity for a cancer associated mucin, MUC1. The recombinant strains of the yeast P. pastoris were developed which secrete single-domain antibody fragment to the culture supernatant as a biologically active protein. Supplementation of medium with sorbitol (in pre-induction phase) and casamino acid or EDTA (in induction phase) provided ideal condition of increasing the yield of VHH production compared to culture condition devoid of above recipe. The secreted protein was purified following a 80% ammonium sulfate precipitation step, followed by a affinity chromatography column. The specific activity in enzyme-linked immunosorbant assay (ELISA) of the purified yeast VHH was higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level and correctly folded production of VHH antibody fragments with potential in vivo diagnostic and therapeutic applications. This is the first report of expression of VHH in P. pastoris.
authors.affiliation: Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, IR Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Rahbarizadeh
authors.forename: Mohammad J
authors.initials: MJ
authors.lastname: Rasaee
authors.forename: Mehdi
authors.initials: M
authors.lastname: Forouzandeh
authors.forename: Abdol-Amir
authors.initials: AA
authors.lastname: Allameh
mesh_terms: D000818:Animals; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology*; D001943:Breast Neoplasms / Q000276:immunology / Q000473:pathology; D002162:Camelus / Q000235:genetics / Q000276:immunology*; D002277:Carcinoma / Q000276:immunology / Q000473:pathology; D045744:Cell Line, Tumor / Q000276:immunology; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000276:immunology / Q000473:pathology; D005260:Female; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors / Q000235:genetics; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D007114:Immunization; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018396:Mucin-1; D009077:Mucins / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D010843:Pichia / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000912:Antibodies, Neoplasm; D000941:Antigens; D000951:Antigens, Neoplasm; D006023:Glycoproteins; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; C060192:MUC1 protein, human; D018396:Mucin-1; D009077:Mucins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins