Effects of signal sequences on the secretion of hen lysozyme by yeast: construction of four secretion cassette vectors.

Abstract

Recombinant protein expression is important in many fields— medicine, biochemistry, molecular biology, etc.—as well as in industry. In general, bacterial, fungal, animal and plant cells have been used for this purpose. In the case of the bacterium Escherichia coli, recombinant proteins often accumulate as inclusion bodies, although expression level is high (Imoto et al., 1987). Therefore, the inclusion body must be renatured into active forms. Moreover, in many cases, methionine is not removed from the N-terminal end of the protein, which may affect the function or structure of the protein (Mine et al., 1997). On the other hand, as is the case for animal and plant cells, the level of expression is generally low (Haynes and Weissmann, 1983; Kaufman et al., 1987; Miyaji et al., 1990). Fungi, especially the yeast Saccharomyces cerevisiae, can secrete foreign protein in an active form and the secretion level is higher relative to that of animal or plant cells. Glycosylation occurs both in yeast as well as in animal cells (Ballou, 1990). Thus, expression in yeast is one of the best ways to acquire and purify large quantities of protein in an active form. Expression and secretion of various mammalian proteins in yeast have been reported: interferon α (Hitzeman et al., 1981), interferon γ (Derynck et al., 1983), interleukin 2 (Shaw et al., 1985) and epidermal growth factor (Brake et al., 1984), Fab (Horwitz et al., 1988). However, recombinant proteins are not always secreted from yeast, which is dependent on the target protein, host strain and secretion–signal sequence. The choice of a suitable secretion–signal sequence for a given protein is an important factor in the final yield of secreted protein. The addition of a secretion–signal sequence onto the mature protein is essential to acquire an active protein. One way to select the signal sequence is to use the original signal sequence of the desired protein gene (Jigami et al., 1986; Nakamura et al., 1986). However, it is limited only to secretory proteins. Alternatively, a yeast signal sequence may be used. In this case, it can be applied not only to secretory proteins but also to non-secretory proteins. Before a foreign protein is expressed in yeast, some trials are required to find out which signal sequence is most suitable. We constructed four ‘secretion cassette vectors’ containing four different yeast secretion signals: αF (Brake, 1990; Hitzeman et al., 1990), KILM1 (Skipper et al., 1985), PHO1 (Laroche et al., 1994) and SUC2 (Hitzeman et al., 1990).