Many therapeutically interesting proteins require
modifications such as disulfide bond-formation apart from folding per
se. As expression-hosts, yeasts combine the ease of manipulation of
microbes with the presence of a secretory pathway that is capable of
mediating such eukaryotic modifications. Kluyveromyces lactis,
amongst other yeasts, is capable of low-level secretion of active
single-chain antibodies without further engineering. To ultimately
identify and elucidate factors and mechanisms that affect folding and
secretion, we constructed a recombinant gene for a c-myc-tagged single-
chain antibody against oxazolone (scFvOx), fused to the mating factor
alpha prepro-sequence. The construct is expressed from the LAC4
promoter and terminated by the TEF1 terminator. It is followed by
the KlGAL80 promoter-controlled LAC4 gene which serves for
integration by homologous recombination and as a reporter in strain
optimization. A selection system based on reconstitution of lactose
growth was developed for integration of the expression cassette in the
LAC4 locus. Expression of scFvOx was induced by lactose and a
product was present in Western Blot of culture supernatants. The
secreted antibody was active for oxazolone-binding in ELISA.