Cloning and expression of human endothelial-monocyte-activating polypeptide 2 (EMAP-2) and identification of its putative precursor.
Endothelial-monocyte-activating polypeptide 2 (EMAP-2) modulates a range of properties of endothelial cells, monocytes and neutrophils in vitro, and induces an acute inflammatory reaction and tumour regression in vivo. We generated the full-length human cDNA sequences of EMAP-2 and its putative precursor pro-EMAP-2 as PCR products. These were cloned into the pCR3 vector and subcloned into pGEX-2T for expression as fusion products with glutathione-S-transferase (GST). Recombinant EMAP-2 (rEMAP-2) was isolated by thrombin cleavage of the fusion protein, followed by affinity chromatography. rEMAP-2 retained biological activity, which was blocked by polyclonal antibodies raised against GST-EMAP-2. By Western blotting, a 34-kDa product corresponding to the predicted precursor proEMAP-2 was detected in lysates of the U937 monocytic cell line, while supernatants contained higher levels of the mature 22-kDa molecule.
Tas
M P
MP
University of Nottingham, Laboratory of Molecular Oncology, Cancer Research Campaign Department of Clinical Oncology, City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK.
Houghton
J
J
Jakobsen
A M
AM
Tolmachova
T
T
Carmichael
J
J
Murray
J C
JC
D000818:Animals; D003001:Cloning, Molecular; D016207:Cytokines; D015870:Gene Expression; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D016601:RNA-Binding Proteins; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
D016207:Cytokines; D009363:Neoplasm Proteins; D011498:Protein Precursors; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C077219:small inducible cytokine subfamily E, member 1