title: High level expression of a promising anti-idiotypic antibody fragment vaccine against HIV-1 in Pichia pastoris.
abstract: We have expressed the anti-idiotypic antibody 3H6 Fab directed against the HIV-1 broadly neutralising antibody 2F5 in methylotrophic yeast Pichia pastoris. The chimeric human/mouse Fab fragment was expressed under control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Bioreactor experiments showed the ability of the recombinant P. pastoris clone to secrete up to 260 mg/L Fab fragment in the culture supernatant during a five days cultivation time. Codon optimisation of the Fab expression cassette gave no further improvement of specific productivity when comparing 12 clones of each construct. The subsequent purification of Fab containing supernatants was done by anion exchange and size-exclusion chromatography with a recovery resulting in 70% of the recombinant protein. For verification of the suitability of the expression system we characterised the expressed protein with respect to both, its specificity and binding affinity and could not detect any significant difference between products from yeast derived and the hybridoma derived product. Finally we tested the implicit requirement of the carbohydrate moiety in the H2 loop of the original 3H6 antibody by introducing an asparagine to alanine replacement and, in a second experiment, inhibition of N-glycosylation by tunicamycin treatment. Biochemical analysis confirmed that the N-glycosylation does not contribute to the binding properties of 3H6.
authors.affiliation: Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Johannes S
authors.initials: JS
authors.lastname: Gach
authors.forename: Michael
authors.initials: M
authors.lastname: Maurer
authors.forename: Rainer
authors.initials: R
authors.lastname: Hahn
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
authors.forename: Hermann
authors.initials: H
authors.lastname: Katinger
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
mesh_terms: D016915:AIDS Vaccines; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors; D006031:Glycosylation; D015497:HIV-1; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D016915:AIDS Vaccines; D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins