Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation

FEBS Letters

Assfalg-Machleidt, Irmgard⁴  Genenger, Gabriele¹  Auerswald, Ennes A.¹  Bode, Wolfram²  Kos, Janko³ 

[1] Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik Innenstadt, Ludwigs-Maximillians-Universität München, Nußbaumstr. 20, D-8000 München 2, FRG;Max-Planck-Institut für Biochemie, D-8033 Martinsried, FRG;Odsek za biokemijo, Institut Jozef Stefan, Univerza E. Kardelja, Jamova 39, YU-61000 Ljubljana, Yugoslavia;Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität München, Goethestr. 33, D-8000 München 2, FRG

关键词: Cysteine proteinase;    Enzyme inhibitor;    Cystatin;    Synthetic gene;    Protein engineering;    (Chicken);    aa;    amino acids;    MS-2 pol;    RNA bacteriophage MS-2 polymerase;    PAGE;    polyacrylamide gel electrophoresis;   

DOI  :  10.1016/0014-5793(89)80127-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown