Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli.
The azurin-encoding azu gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. A purification procedure was developed to isolate the azurin obtained from the E. coli cells. No differences were observed between azurins isolated from P. aeruginosa and E. coli. A non-reconstitutable azurin-like protein, azurin*, with a spectral ratio (A625/A280) less than 0.01 could be separated from holo-azurin with a spectral ratio of 0.58 (+/- 0.01). The properties of azurin* were examined by electrophoretic (SDS-PAGE and IEF) and spectroscopic (UV/vis, 1H-NMR, static and dynamic fluorescence) techniques, and compared to the properties of holo-azurin and apo-azurin. Azurin* resembles apo-azurin (same pKa* values of His-35 and His-117, same fluorescence characteristics). However, it has lost the ability to bind Cu-ions. It is tentatively concluded that azurin* is a chemically modified form of azurin, the modification possibly being due to oxidation of the ligand residue Cys-112 or the formation of a chemical bond between the ligand residues Cys-112 and His-117. In agreement with previous results from Hutnik and Szabo (Biochemistry (1989) 28, 3923-3934), fluorescence experiments show that the heterogeneous fluorescence decay observed for holo-azurin is not due to the presence of azurin*, but most likely originates from conformational heterogeneity of the holo-azurin.
van de Kamp
M
M
Chemistry Department, Gorlaeus Laboratories, Leiden University, The Netherlands.
Hali
F C
FC
Rosato
N
N
Agro
A F
AF
Canters
G W
GW
D001400:Azurin / Q000096:biosynthesis / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007525:Isoelectric Focusing; D009682:Magnetic Resonance Spectroscopy; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011995:Recombination, Genetic; D013050:Spectrometry, Fluorescence
D001400:Azurin