cDNA cloning, bacterial expression, in vitro renaturation and affinity purification of the zinc endopeptidase astacin.
Astacin (EC 3.4.24.21) from the freshwater crayfish (Astacus astacus) is a prototype for the metzincin superfamily and for the astacin family of zinc peptidases, enzymes which are involved in hatching processes, embryonic patterning and tissue remodelling. Here we report on the cloning and overexpression in Escherichia coli of an astacin cDNA which was reverse-transcribed from crayfish midgut-gland mRNA. A cDNA construct based on this clone was generated which comprised the nucleotide sequence encoding mature astacin devoid of the signal and propeptide. This construct was cloned into the pET3a vector and used to transform E. coli BL21(DE3) cells. Recombinant astacin was purified from inclusion bodies and dissolved under reducing conditions. For folding, the protein was diluted into neutral buffer containing l-arginine, GSH and EDTA. Eventually, Zn(2+) was added by dialysis and the fraction of active enzyme was affinity-purified on immobilized Pro-Leu-Gly hydroxamate. As shown by superimposition of the corresponding three-dimensional structures, this inhibitor binds to a region of the active-site cleft that is conserved in most metzincins. Therefore this principle behind this affinity technique, originally introduced for fibroblast collagenase by Moore and Spilburg [Biochemistry (1986) 25, 5189-5195], is applicable throughout the metzincin superfamily of metalloproteases, despite their otherwise differing cleavage specificities. Recombinant astacin is active on gelatine zymograms and in a quenched fluorescence assay, yielding kinetic parameters comparable with those of wild-type astacin purified from crayfish stomach.
Reyda
S
S
Zoologisches Institut, Universität Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany.
Jacob
E
E
Zwilling
R
R
Stöcker
W
W
D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D017510:Protein Folding; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015032:Zinc / Q000737:chemistry
D009842:Oligopeptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C090221:prolyl-leucyl-glycine hydroxamate; D008666:Metalloendopeptidases; C071288:astacin; D015032:Zinc