Expression and purification of recombinant vascular endothelial-statin.
We have recently discovered VE-statin (egfl7), a novel protein specifically expressed by endothelial cells during mouse development and in the adult. VE-statin is expected to play an essential role during the formation of new blood vessels. Here, we have expressed the coding sequence for VE-statin in bacteria and designed a protocol for the production and purification of large amounts of the protein. The protocol is based on an affinity purification step followed by a strong cation-exchange chromatography and a refolding procedure adapted to epidermal growth factor domains and also to the high tendency of the protein to precipitate in concentrated solutions. The purified protein is pure as assessed by SDS-PAGE, immunoblot and mass spectrometry, and is biologically active as a repressor of human aortic smooth muscle cell migration induced by platelet-derived growth factor-BB.
Caetano
Bertrand
B
CNRS UMR8526, Université de Lille II, Institut Pasteur de Lille, 1 rue Calmette, 59021 Lille Cedex, France.
Drobecq
Hervé
H
Soncin
Fabrice
F
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D000077214:Becaplermin; D002135:Calcium-Binding Proteins; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D066255:EGF Family of Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D066255:EGF Family of Proteins; C479627:Egfl7 protein, mouse; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin