PROTEIN-PURIFICATION, GENE CLONING AND SEQUENCING OF AN ACIDIC ENDOPROTEASE FROM MYXOCOCCUS-XANTHUS DK101

An acidic endoprotease (MAEP) secreted during vegetative growth by Myx ococcus xanthus DK101 was purified to homogeneity by a series of chrom atographic procedures. The endoprotease cleaved the Phe-Met bond of Ic -casein under acidic conditions (pH 5.9). Its apparent molecular mass and its isoelectric point have been estimated to be 12 kDa and 4.5, re spectively. From the N-terminal amino acid sequence, a set of two prim ers for polymerase chain reaction have been designed. Amplification of the corresponding DNA fragment (84 bp) generated a probe, then used t o screen an expression DNA library of M. xanthus and to isolate a reco mbinant. plasmid which contained a 2127-bp insert. The nucleotide sequ ence included an open reading frame (ORF) of 585 nucleotides, encoding 195 amino acids, that exhibited a high degree of similarity with the N-terminal amino acid sequence of the purified MAEP. The polypeptide s equence inferred from this ORF revealed that the mature enzyme should contain 131 amino acids arising from a 195-amino-acid precursor protei n.