A three-step purification method of large quantities of human recombinant alpha endothelial cellular growth factor for clinical use.
The endothelial cellular growth factor alpha-ECGF is a candidate drug for the induction of therapeutic neoangiogenesis. Its use in extensive experimental and clinical trials is hampered by the fact that currently published purification procedures allow only small yields, and the absence of pyrogenic impurities is not demonstrated. The rh alpha-ECGF was expressed in E. coli. Isolation of rh alpha-ECGF from E. coli lysates to apparent homogenicity was achieved by a three step purification procedure involving ionic exchange, heparin-sepharose and polymyxin B chromatography. By this method, 200 mg of rh alpha-ECGF was purified from 15 g wet weight E. coli bacteria. The isolated protein of 18 kDa appeared as a single band after SDS gel electrophoresis and subsequent silver-staining. The biological activity was expressed in the chorion-allantois-membrane assay and in the 3H-thymidine proliferation in baby hamster kidney cells. Drug trials with rabbits revealed no increase in body temperature after intravenous injections with 1 mg rh-ECGF.
Sauter
Alexander
A
Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, D-68135 Mannheim, Germany. alexander.sauter@hno.ma.uni-heidelberg.de
Lambert
Katherine L
KL
Rupf
Ann-Katrin
AK
Von Specht
Bernd-Ulrich
BU
Hörmann
Karl
K
Naim
Ramin
R
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D016228:Endothelial Growth Factors / Q000302:isolation & purification* / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008566:Membranes / Q000187:drug effects*; D010063:Ovum / Q000737:chemistry; D011112:Polymyxin B / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D014169:Transformation, Bacterial
D016228:Endothelial Growth Factors; C037405:heparin-sepharose; D004247:DNA; D012685:Sepharose; D011112:Polymyxin B