Expression, purification, biochemical and pharmacological characterization of a recombinant aprotinin variant
Date
2004
Authors
Apeler H.
Peters J.
Schroder W.
Schneider K.-H.
Lemm G.
Hinz V.
Rossouw G.J.
Dembowsky K.
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Aprotinin (CAS 9087-70-1) is known as a potent inhibitor of serine proteases such as trypsin, plasmin, tissue and plasma kallikrein. In this study, an aprotinin variant was designed by means of rationale mutagenesis that differs from aprotinin by two amino acids in the active site and by seven amino acids in the backbone. The recombinant protein is expressed in a secretory yeast system enabling large scale production. A purification procedure was developed to yield high amounts of pure and correctly processed aprotinin variant. The changes in the active site of the aprotinin variant increase the potency towards inhibition of plasma kallikrein whereas the inhibition of plasmin is only marginally reduced. The net charge of the molecule is reduced from the basic (IP 10.5) to the neutral range (IP 5.6). The recombinant aprotinin variant shows a decrease of immunogenicity in several models. No cross-reactivity with human and rabbit antibodies directed against aprotinin was observed both in in vivo and in ex vivo studies. In addition, the variant is more potent in a rat brain edema model of acute subdural hematoma compared to aprotinin.
Description
Keywords
antiinflammatory agent, aprotinin, aprotinin variant, kallikrein, plasmin, recombinant protein, serine proteinase inhibitor, unclassified drug, amino acid sequence, animal experiment, animal model, animal tissue, article, brain edema, brain water, chimpanzee, controlled study, cross reaction, dog, drug potency, drug purification, enzyme active site, enzyme inhibition, ex vivo study, female, human, human cell, immunogenicity, in vitro study, in vivo study, inhibition kinetics, male, nonhuman, protein expression, rabbit, rat, reflex, Saccharomyces cerevisiae, site directed mutagenesis, subdural hematoma, surface charge, Amino Acids, Animals, Aprotinin, Body Water, Brain Chemistry, Brain Edema, Chemistry, Physical, Chromatography, High Pressure Liquid, Cloning, Molecular, Cross Reactions, DNA, Complementary, Dogs, Electrophoresis, Capillary, Electrophoresis, Polyacrylamide Gel, Female, Fermentation, Freeze Drying, Hand Strength, Hemodynamic Processes, Histamine Release, Isoelectric Focusing, Male, Molecular Weight, Pan troglodytes, Peptide Mapping, Protease Inhibitors, Rats, Rats, Wistar, Recombinant Proteins, Saccharomyces cerevisiae, Sequence Analysis, Protein
Citation
Arzneimittel-Forschung/Drug Research
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54
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