Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli.
Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.
Arcone
R
R
CEINGE, Biotecnologie Avanzate, Napoli, Italy.
Arpaia
G
G
Ruoppolo
M
M
Malorni
A
A
Pucci
P
P
Marino
G
G
Ialenti
A
A
Di Rosa
M
M
Ciliberto
G
G
D000595:Amino Acid Sequence; D017305:Annexin A1 / Q000737:chemistry* / Q000494:pharmacology; D001483:Base Sequence; D001681:Biological Assay; D003001:Cloning, Molecular; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology
D017305:Annexin A1; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2