Purification, crystallization and preliminary X-ray diffraction analysis of recombinant human neutrophil-activating peptide 2 (rhNAP-2).
The potent activator and chemoattractant for human neutrophils, neutrophil-activating peptide 2 (NAP-2), has been cloned and expressed in Escherichia coli. The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC. The biological activity of recombinant human NAP-2 (rhNAP-2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP-2. rhNAP-2 has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P222 with unit cell dimensions of a = 30.8 A, b = 39.5 A and c = 95.3 A. A packing density of 3.8 A3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed. The crystals were shown to diffract to beyond 2.0 A on a conventional X-ray source. They are stable to X-rays for several days and are thus suitable for high resolution structure determination.
Kungl
A J
AJ
Sandoz Research Institute, Wien, Austria.
Machius
M
M
Huber
R
R
Schwer
C
C
Lam
C
C
Aschauer
H
H
Ehn
G
G
Lindley
I J
IJ
Auer
M
M
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D019272:Leukocyte Elastase; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology; D010196:Pancreatic Elastase / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D001620:beta-Thromboglobulin
C061615:PPBP protein, human; D010455:Peptides; D011994:Recombinant Proteins; D001620:beta-Thromboglobulin; C009862:connective tissue-activating peptide; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase