High levels of inducible expression of cloned β-galactosidase ofKluyveromyces lactis inSaccharomyces cerevisiae (original) (raw)

1986, Applied Microbiology and Biotechnology

The LAC4 gene of Kluyveromyces lactis CBS2360 coding for fl-galactosidase was isolated from a K. lactis gene bank. The gene was then cloned in a yeast expression vector pBCL26, derived from pLG2 (Guarente 1983), under the control of the inducible GALl-10 UAS/CYC1 yeast hybrid promoter. Two constructions were obtained, pBCLG2 and pBCLG4, that bear the LAC4 gene in the two opposite orientations and we have studied the expression of K. lactis fl-galactosidase in yeast cells transformed with these two plasmids and under different growth conditions. High levels of expression induced by galactose were observed with pBCLG2, which bears the LAC4 gene in the correct orientation, while a low constitutive level of expression was observed in pBCLG4 transformants both in glucose and in galactose media. The expression of the heterologous protein, under induced conditions, appears to be strongly influenced by the growth phase of the culture, with a sharp increase of the specific activity of the enzyme and of its level, calculated as percent of total protein, at the beginning of the stationary phase of growth, during which time the fl-galactosidase reaches a level of 15% of total cellular protein. A. Velati-Bellini et al.: High expression of fl-galactosidase in budding yeast 125

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