Rapid purification of the Aeromonas proteolytica aminopeptidase: crystallization and preliminary X-ray data.
The heat-stable aminopeptidase from Aeromonas proteolytica has been purified using two new procedures, with the aim of preparing large single crystals for X-ray analysis. In a first procedure, we tried to avoid any drastic conditions capable of inducing microheterogeneities in the protein sample. The enzyme was purified through two chromatographic steps based on hydrophobic interactions and ion exchange. In a second procedure a heat treatment of the protein to a temperature of 70 degrees C over 5 to 8 h was performed. Both procedures led to an electrophoretically homogeneous and crystallizable aminopeptidase; however, unexpectedly, the crystals obtained through the first procedure contained, in addition to the native aminopeptidase, a cleaved form of the enzyme which has been characterized. Only the native protein was present when the second procedure was used. Large crystals obtained with the native protein form, having an approximate size of 0.4 x 0.4 x 0.6 mm, produced an X-ray diffraction pattern that exhibited the symmetry associated with the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22). The unit cell parameters were a = 109.1 A and c = 97.8 A. Assuming one molecule/asymmetric unit, a value of VM = 2.6 A3/Da and an approximate solvent content of 45% could be estimated. Measurable diffraction intensities were observed at a resolution of 2.5 A.
Schalk
C
C
Marion Merrell Dow Research Institute, Strasbourg, France.
Remy
J M
JM
Chevrier
B
B
Moras
D
D
Tarnus
C
C
D000333:Aeromonas / Q000201:enzymology*; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D003460:Crystallization; D006358:Hot Temperature; D008969:Molecular Sequence Data; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D000626:Aminopeptidases; C038222:bacterial leucyl aminopeptidase