Design of an aprotinin variant with inhibitory activity against chymotrypsin and cathepsin G by recombinant DNA technology.
The [Phe15,17, Glu52]aprotinin gene was constructed from synthetic [Glu52]aprotinin gene by DNA cassette mutagenesis. For substitution of the P1, P'1, P'2 residues of aprotinin (Lys15-Ala16-Arg17) two complementary synthetic oligonucleotides including a new segment coding for the Phe15-Ala16-Phe17 sequence were inserted into the expression vector. The expression was carried out in an E. coli host and [Phe15,17, Glu52]aprotinin was obtained as fusion protein with bacteriophage MS2 polymerase and deposited in inclusion bodies. After CNBr cleavage and renaturation of aprotinin on CM Sepharose Fast Flow the active aprotinin variant was isolated and its specificities investigated. The new [Phe15,17, Glu52]aprotinin was shown to be an inhibitor for bovine chymotrypsin (Ki = 6.5 x 10(-10)M) essentially identical to the previously prepared semisynthetic [Phe15]aprotinin (Ki = 2.0 x 10(-10)M) (1). However, the new variant exhibited an additional inhibitory activity against cathepsin G from human leukocytes (Ki = 5.0 x 10(-7)M) in contrast to semisynthetic [Phe15] variant which was inactive.
Brinkmann
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Fakultät für Chemie, Universität Bielefeld, FRG.
Tschesche
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D000595:Amino Acid Sequence; D007611:Aprotinin / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D001483:Base Sequence; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004274:DNA, Recombinant; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009154:Mutation; D012697:Serine Endopeptidases
D004274:DNA, Recombinant; D007611:Aprotinin; D004259:DNA-Directed DNA Polymerase; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G