Purification and characterization of active recombinant rat kallikrein rK9.
The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.
Zani
M
M
Laboratory of Enzymology and Protein Chemistry, INSERM EMI-U 00-10, University François Rabelais, France.
Brillard-Bourdet
M
M
Lazure
C
C
Juliano
L
L
Courty
Y
Y
Gauthier
F
F
Moreau
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D007610:Kallikreins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008297:Male; D010843:Pichia / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D012661:Semen / Q000737:chemistry / Q000378:metabolism; D012669:Seminal Vesicles / Q000378:metabolism; D013379:Substrate Specificity; D020840:Tissue Kallikreins / Q000737:chemistry
D011506:Proteins; D011994:Recombinant Proteins; D007610:Kallikreins; D020840:Tissue Kallikreins; D017430:Prostate-Specific Antigen