Open AccessProtein purification, gene cloning and sequencing of an acidic endoprotease from Myxococcus xanthus DK101
Author(s) -
LUCAS Nathalie,
MAZAUDAUJARD Catherine,
BREMAUD Laure,
CENATIEMPO Yves,
JULIEN Raymond
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18863.x
Subject(s) - myxococcus xanthus , biochemistry , amino acid , peptide sequence , nucleic acid sequence , open reading frame , biology , microbiology and biotechnology , isoelectric point , plasmid , molecular cloning , gene , enzyme , mutant
An acidic endoprotease (MAEP) secreted during vegetative growth by Myxococcus xanthus DK101 was purified to homogeneity by a series of chromatographic procedures. The endoprotease cleaved the Phe‐Met bond of K ‐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, respectively. From the N‐terminal amino acid sequence, a set of two primers for polymerase chain reaction have been designed. Amplification of the corresponding DNA fragment (84 bp) generated a probe, then used to screen an expression DNA library of M. xanthus and to isolate a recombinant plasmid which contained a 2127‐bp insert. The nucleotide sequence 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 sequence inferred from this ORF revealed that the mature enzyme should contain 131 amino acids arising from a 195‐amino‐acid precursor protein.