Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system.
A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN-III-ompA Escherichia coli expression system. After osmotic shock of the E. coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S-carboxymethylpapain affinity chromatography. The resulting inhibitory material was characterized by SDS/PAGE, reversed-phase HPLC, peptide mapping and amino acid sequencing. The recombinant variant chicken AEF-[S1----M, M29----I, M89----L]cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin. The purified variant showed a native-chicken-cystatin-like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N-labelled recombinant inhibitor were compared with those of the natural inhibitor.
Auerswald
E A
EA
Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgischen Klinik Innenstadt, Universität München, Federal Republic of Germany.
Genenger
G
G
Mentele
R
R
Lenzen
S
S
Assfalg-Machleidt
I
I
Mitschang
L
L
Oschkinat
H
H
Fritz
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D015891:Cystatins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000096:biosynthesis
D015891:Cystatins; D011994:Recombinant Proteins; C100158:cystatin, egg-white