Comparison of natural and recombinant clitocypins, the fungal cysteine protease inhibitors.
A member of the cysteine protease inhibitor clitocypin gene family from basidiomycete Clitocybe nebularis was expressed in Escherichia coli. Following careful optimization of the expression procedure the active inhibitor was purified from inclusion bodies and its properties examined and compared to those of the natural clitocypin. The CD spectrum of recombinant clitocypin was similar to that of natural clitocypin, indicating that protein was properly refolded during purification. In spite of some differences in primary structure, structural, functional and immunological equivalence was established. Kinetic analyses of the natural and recombinant clitocypins were performed. Both clitocypins inhibited a range of cysteine proteases to a similar extent, and demonstrated an unusually broad inhibitory spectrum, including distantly related proteases, such as papain and legumain, belonging to different protease families. The homogenous, biologically active recombinant clitocypin is obtained at levels adequate for further structure-function studies.
Sabotic
Jerica
J
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
Galesa
Katja
K
Popovic
Tatjana
T
Leonardi
Adrijana
A
Brzin
Joze
J
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D001487:Basidiomycota / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D015151:Immunoblotting; D002479:Inclusion Bodies / Q000737:chemistry; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D000906:Antibodies; C411656:Clitocypin protein, Clitocybe nebularis; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins