LEKTI domain 15 is a functional Kazal-type proteinase inhibitor.
The multidomain proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor) consists of 15 potential serine proteinase inhibitory domains. In various diseases such as the severe skin disorder Netherton syndrome as well as atopy, defects in the gene encoding LEKTI have been identified that generate premature termination codons of translation, suggesting a specific role of the COOH-terminal part of LEKTI in healthy individuals. We overexpressed and purified a sequence comprising the 15th domain of LEKTI for further characterisation. Here, we present a high yield expression system for recombinant production and efficient purification of LEKTI domain 15 as a highly soluble protein with a uniform disulfide pattern that is identical to that of other known Kazal-type inhibitors. Also, the expected P1P1' site was confirmed. LEKTI domain 15 is a well-structured protein as verified by circular dichroism (CD) spectroscopy and a tight-binding and stable inhibitor of the serine proteinase trypsin. These findings confirm the designation of domain 15 as a proteinase inhibitor of the Kazal family.
Vitzithum
Klaus
K
Lehrstuhl für Biopolymere, Universität Bayreuth, Universitätstrasse 30, D-95447 Bayreuth, Germany.
Lauber
Thomas
T
Kreutzmann
Peter
P
Schulz
Axel
A
Sommerhoff
Christian P
CP
Rösch
Paul
P
Marx
Ute C
UC
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018076:DNA, Complementary; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053491:Proteinase Inhibitory Proteins, Secretory / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014169:Transformation, Bacterial; D014357:Trypsin / Q000378:metabolism
D018076:DNA, Complementary; D004220:Disulfides; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D015842:Serine Proteinase Inhibitors; D014357:Trypsin