Processing, disulfide pattern, and biological activity of a sugar beet defensin, AX2, expressed in Pichia pastoris

AX2 is a 46-amino-acid cysteine-rich peptide isolated from sugar beet leave s infected with the fungus Cercospora beticola (Sacc). AX2 strongly inhibit s the growth of C. beticola and other filamentous fungi, but has little or no effect against bacteria. AX2 is produced in very low amounts in sugar be et leaves, and to study the protein in greater detail with respect to biolo gical function and protein structural analysis, the methylotrophic yeast Pi chia pastoris was used for large-scale production. The amino acid sequence, processing of the signal peptide, disulfide bridges, and biological activi ty of the recombinant protein were determined and compared with that of the authentic AX2. In P. pastoris, the protein was expressed with an additiona l N-terminal arginine. The disulfide bonding was found to be identical to t hat of the authentic AX2. However, when tested in in vitro bioassay, the bi ological activity of the recombinant protein was slightly lower than that m easured for the authentic protein. Furthermore, the recombinant protein was significantly more sensitive to Ca2+ than the authentic protein. This is m ost probably due to the extra arginine, since no other differences between the two proteins have been found. (C) 1999 Academic Press.