Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system

Expression systems of human and silkworm lysozymes were constructed using t he methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of alpha -factor (a peptide pheromone derived from yeast ) and the native signal sequences of these lysozymes, were used as secretio n signals. When the alpha -factor leader is used as the signal sequence, hu man lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is se creted more efficiently than human lysozyme. Therefore, we expected that hu man lysozyme cDNA with a silkworm native signal would be secreted more effi ciently than human lysozyme with its native signal. However, its level of e xpression was not increased. This result indicates that the native signal o f silkworm lysozyme does not promote the secretion of the lysozyme, but rat her alpha -factor leader inhibits the secretion. Silkworm lysozyme with the alpha -factor leader is so unstable that it could be easily attacked by so me proteases and our findings suggest that the level of expression of heter ologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.