Expression, purification, biochemical and pharmacological characterization of a recombinant aprotinin variant.
Aprotinin (GAS 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.
Pharma Operations Biotechnology, Bayer HealthCare AG Pharma, Wuppertal, Germany. Heiner.Apeler@bayerhealthcare.com
Heiner
H
Apeler
Jörg
J
Peters
Werner
W
Schröder
Karl-Heinz
KH
Schneider
Georg
G
Lemm
Volker
V
Hinz
Gawie J
GJ
Rossouw
Klaus
K
Dembowsky
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D007611:Aprotinin / Q000096:biosynthesis* / Q000276:immunology / Q000494:pharmacology*; D001834:Body Water / Q000378:metabolism; D001923:Brain Chemistry / Q000187:drug effects; D001929:Brain Edema / Q000188:drug therapy; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003429:Cross Reactions; D018076:DNA, Complementary / Q000096:biosynthesis; D004285:Dogs; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005285:Fermentation; D005612:Freeze Drying; D018737:Hand Strength / Q000502:physiology; D006439:Hemodynamics / Q000187:drug effects; D006636:Histamine Release / Q000187:drug effects; D007525:Isoelectric Focusing; D008297:Male; D008970:Molecular Weight; D002679:Pan troglodytes / Q000276:immunology; D010449:Peptide Mapping; D011480:Protease Inhibitors / Q000276:immunology / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D020539:Sequence Analysis, Protein
D000596:Amino Acids; D018076:DNA, Complementary; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D007611:Aprotinin