Protein purification, gene cloning and sequencing of an acidic endoprotease from Myxococcus xanthus DK101.
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 kappa-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.
Lucas
N
N
Institut de Biotechnologie, Faculté des Sciences, Limoges, France.
Mazaud-Aujard
C
C
Bremaud
L
L
Cenatiempo
Y
Y
Julien
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016941:Myxococcus xanthus / Q000201:enzymology* / Q000235:genetics*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D010450:Endopeptidases; C088186:Myxococcus acidic endoprotease