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Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation
Author(s) -
Auerswald Ennes A.,
Genenger Gabriele,
Assfalg-Machleidt Irmgard,
Kos Janko,
Bode Wolfram
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80127-9
Subject(s) - cystatin , fusion protein , microbiology and biotechnology , recombinant dna , papain , expression vector , gene , biology , cathepsin l , cathepsin h , fusion gene , chemistry , escherichia coli , biochemistry , cystatin c , cysteine , enzyme , renal function
A synthetic gene coding for the cysteine proteinase inhibitor (desSer 1 Ile29 Leu89) chicken cystatin was cloned and expressed in E. coli . The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8. Expression as fusion protein was performed in a temperature‐inducible E. coli system. The expression product was synthesized as 20% of total E. coli protein. The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N‐terminal sequence confirmed up to position 37. The properties of the purified material correspond to those of natural chicken cystatin. The recombinant cystatin variant binds anti‐chicken cystatin IgG, is inhibitorily active and displays K i values with papain and with cathepsin B similar to those determined for natural chicken cystatin.