Human interferon-beta, expressed in Saccharomyces cerevisiae, is predominantly directed to the vacuoles. Influence of modified co-expression of secretion factors and chaperones.
Expression of the human interferon-beta (hIFN-beta) gene was found to be very toxic for Saccharomyces cerevisiae. An integrative expression cassette, containing the hIFN-beta gene under control of the inducible galactokinase (GAL1) promoter in combination with the alpha-factor prepro-secretion signal, was used to study the secretion process in more detail. Specific differences were found between a vacuolar proteinase--mutant and a normal laboratory yeast strain. Cell organelle fractionation, carried out with the recombinant C13-ABYS66 strain, revealed that 99% of the hIFN-beta remained intracellular and that the majority was associated with the vacuolar fraction. The secretion efficiency in the latter strain was investigated by overexpressing chaperone molecules (HSP70 and BiP) and homologous secretion factors (SEC1 and SEC18). Only the presence of HSP70 resulted in a 5-fold increase in secreted hIFN-beta.
Demolder
J
J
Laboratory of Molecular Biology, Gent University, Belgium.
Fiers
W
W
Contreras
R
R
D000251:Adenosine Triphosphatases; D001483:Base Sequence; D005656:Fungal Proteins / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D016899:Interferon-beta / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; C497931:SEC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D016899:Interferon-beta; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae