Recombinant production, purification and biochemical characterization of domain 6 of LEKTI: a temporary Kazal-type-related serine proteinase inhibitor.
Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a 15-domain serine proteinase inhibitor which is of pathophysiological relevance for skin diseases and atopy. Domains 2 and 15 of LEKTI contain six cysteine residues and match the Kazal-type inhibitor motif almost exactly. The other 13 domains seem to be Kazal-type derived but lack the cysteines in positions 3 and 6 usually conserved within this family of inhibitors. Here, we report the recombinant production and comprehensive biochemical characterization of the 7.7 kDa LEKTI domain 6 (LD-6). Testing a selected number of different serine proteinases, we show that both native and recombinant LD-6 exhibit a significant but temporary inhibitory activity on trypsin. Furthermore, the relation of LEKTI domain 6 to Kazal-type inhibitors is confirmed by determining its disulfide bond pattern (1-4/2-3) and its P(1) site located after the second Cys residue of LD-6. The established strategy for the recombinant production of LEKTI domain 6 will enable further investigation of its mode of action and its physiological role.
Kreutzmann
Peter
P
IPF PharmaCeuticals GmbH, Feodor-Lynen-Strasse 31, D-30625 Hannover, Germany.
Schulz
Axel
A
Ständker
Ludger
L
Forssmann
Wolf-Georg
WG
Mägert
Hans-Jürgen
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D000074163:Serine Peptidase Inhibitor Kazal-Type 5
D002352:Carrier Proteins; D017931:DNA Primers; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5