Secretion, purification and activity of two recombinant pepper endo-beta-1,4-glucanases expressed in the yeast Pichia pastoris.

Two pepper endo-beta-1,4-glucanases, involved in fruit softening (cCel1) and leaf and flower abscission (cCel2), have been expressed in Pichia pastor is. Secretion was obtained by using either the mouse a-factor signal(cCel1) or the native signal sequence (cCel2), Times for optimal expression of the two proteins were different and cCel2 appeared very sensitive to proteolytic degradation, A one-step purification protocol yielded cCel2 in a pure form, while an additional chromatography step was necessary to purify cCel1, The two recombinant proteins are highly active and able to degrade carboxymethylcellulose in viscometric assays. Moreover, they have both a molecular mass (54 kDa) and an isoelectric point (7.2 for cCel2, and 8.5 for cCel1) equal to those of the native proteins, thus suggesting that post-translational modifications have properly occurred. (C) 1998 Federation of European Biochemical Societies.