| Abstrakt: |
The LAC4 gene ofKluyveromyces lactis CBS2360 coding for ß-galactosidase was isolated from aK. lactis gene bank. The gene was when cloned in a yeast expression vector pBCL26, derived from pLG2 (Guarente 1983), under the control of the inducible GAL1-10 USA/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 ofK. lactis ß-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 ß-galactosidase reaches a level of 15% of total cellular protein. |
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