Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation.
A synthetic gene coding for the cysteine proteinase inhibitor (desSer1 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 Ki values with papain and with cathepsin B similar to those determined for natural chicken cystatin.
Auerswald
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EA
Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik Innenstadt, Ludwigs-Maximillians-Universität München, FRG.
Genenger
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G
Assfalg-Machleidt
I
I
Kos
J
J
Bode
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D015891:Cystatins; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D015891:Cystatins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C100158:cystatin, egg-white