Production and structural characterization of amino terminally histidine tagged human oncostatin M in E. coli.
Oncostatin M is a cytokine that acts as a growth regulator on a wide variety of cells and has diverse biological activities including acute phase protein induction, LDL receptor up-regulation and cell-specific gene expression. In order to gather information about the Onc M structure, we established a protocol for large scale production and single step purification of this functional cytokine from bacterial cells. The cDNA of human Onc M was cloned by RT-PCR from total RNA of PMA induced U937 cells. After the addition of a six histidine tag at the N-terminus, the coding region of mature Onc M was cloned in the pT7.7 expression vector. Histidine tagged Onc M was overexpressed in bacterial cells and purified to homogeneity in one step on a metal chelating column. We found that recombinant 6xHis-OncM remains fully active in a growth inhibition assay. Structural characterization of the purified protein was performed by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry. Thermal and pH stability dependence of Onc M was assessed by circular dichroism spectroscopy; the helical content is about 50%, in agreement with the four helix bundle fold postulated for cytokines that bind haematopoietic receptors of type I.
Sporeno
E
E
IRBM (Istituto di Ricerche di Biologia Molecolare) Pietro Angeletti, Pomezia, Rome, Italy.
Barbato
G
G
Graziani
R
R
Pucci
P
P
Nitti
G
G
Paonessa
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods; D016207:Cytokines / Q000096:biosynthesis; D004926:Escherichia coli; D015870:Gene Expression; D006131:Growth Inhibitors / Q000096:biosynthesis*; D006639:Histidine; D006801:Humans; D008961:Models, Structural; D008969:Molecular Sequence Data; D053683:Oncostatin M; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D016207:Cytokines; D006131:Growth Inhibitors; C508351:OSM protein, human; D010455:Peptides; D011994:Recombinant Proteins; D053683:Oncostatin M; D006639:Histidine; D013755:Tetradecanoylphorbol Acetate