Comparison of natural and recombinant clitocypins, the fungal cysteine protease inhibitors
- 作者：Jerica Sabotič ; Katja Galeš ; a ; Tatjana Popovič ; Adrijana Leonardi ; Jož ; e Brzin
- 关键词：Clitocybe nebularis ; Mushroom ; Basidiomycete ; Clitocypin ; Cysteine protease inhibitor ; Heterologous expression ; Cysteine protease ; Cathepsins ; Legumain ; Escherichia coli
- 刊名：Protein Expression and Purification
- 出版年：2007
- 期刊代码：184_10465928
- 类别：bio
- 出版时间：May 2007
- 卷：53
- 期：1
- 页码：104-111
- 文件大小：471 K
摘要
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.