High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris.
In this report, we describe the high-yield secretory expression ( approximately 40 mg x l(-1)) of pure, atrazine-specific Fab fragments (K411B) from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of the alcohol oxidase promoter) into the genome of the yeast cells. Antibody-expressing clones were selected by SDS-PAGE and ELISA and fed-batch fermentations were carried out in a 5-l scale. Both chains of the Fab were successfully expressed upon methanol induction and almost no other proteins were secreted into the media. Approximately 30% of the two chains formed the active Fab fragment containing the intermolecular disulphide bond, as determined by Western blot analysis under non-reducing conditions. Crude culture supernatant was used to study the binding properties of the Fab fragment toward different s-triazines by means of competitive ELISA: the IC50 value for the detection of atrazine was determined from the standard curve as 3 microg x l(-1), which is one magnitude higher than the value obtained with the parental mAb K4E7 but equals that obtained when the same Fab fragment was expressed in Escherichia coli cells. In addition, the cross-reactivity pattern of the Fab from Pichia is comparable to that of E. coli and to the parental mAb K4E7.
Lange
S
S
Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, D-70569, Stuttgart, Germany.
Schmitt
J
J
Schmid
R D
RD
D001280:Atrazine / Q000276:immunology*; D019149:Bioreactors; D004785:Environmental Pollutants / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005506:Food Contamination; D006540:Herbicides / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D000432:Methanol / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis*
D004785:Environmental Pollutants; D006540:Herbicides; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D001280:Atrazine; D000432:Methanol