A rapid and efficient purification method for recombinant annexin V for biophysical studies.
Annexin V binds in a calcium-dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X-ray crystallography and electron microscopy in order to understand the structure-function relationships of the ion channel activity. We describe here a method to obtain very pure recombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium-mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion-exchange chromatography and elutes as a single peak free of any detectable contaminants.
Burger
A
A
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Berendes
R
R
Voges
D
D
Huber
R
R
Demange
P
P
D000818:Animals; D017304:Annexin A5 / Q000302:isolation & purification*; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002473:Cell Wall / Q000737:chemistry; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D008081:Liposomes / Q000378:metabolism; D009997:Osmotic Pressure; D011994:Recombinant Proteins / Q000302:isolation & purification; D014961:X-Ray Diffraction
D017304:Annexin A5; D008081:Liposomes; D011994:Recombinant Proteins; D002118:Calcium