The M20 IL-1 inhibitor. I. Purification by preparative isoelectric focusing in free solution.
An interleukin-1 (IL-1) inhibitor produced by the M20 myelomonocytic cell line has been shown to be active in various in vitro and in vivo IL-1 induced parameters. This inhibitor has been purified from the conditioned medium by gel filtration through a Sephacryl S-300 column or dye ligand chromatography on Affi-Gel blue column, followed by isoelectric focusing in free solution in the pH range 3-5 using the Rotofor cell. When gel filtration by FPLC with the Superose 12 column was used as the final step, the combined sequence of purification procedures resulted in a 1600-fold purification of the IL-1 inhibitor. The purified IL-1 inhibitor has a molecular weight of approximately 52 +/- 4 kDa and a pI of 4.15 +/- 0.1. By SDS-PAGE analysis the inhibitor preparation thus obtained showed the presence of two protein bands, while a few closely spaced protein bands were seen by analytical isoelectric focusing in polyacrylamide gels (pH 3-6). Some of these bands in PAGIF might correspond to different degrees of glycosylation of the inhibitory protein. Although the M20 IL-1 inhibitor has not yet been purified to homogeneity, it should be stressed that the procedures used, allowed us to remove the great majority of the proteins present in the medium in which the M20 cells were cultured, and to recover in satisfactory yield the inhibitor which we consider likely to be present in the conditioned medium in subnanomolar concentrations.
Peritt
D
D
Oncology Department, Hadassah University Hospital, Jerusalem, Israel.
Flechner
I
I
Okunev
E
E
Yanai
P
P
Halperin
T
T
Treves
A J
AJ
Barak
V
V
D002460:Cell Line; D003470:Culture Media; D016207:Cytokines / Q000502:physiology*; D006801:Humans; D007375:Interleukin-1 / Q000037:antagonists & inhibitors*; D007526:Isoelectric Point; D008970:Molecular Weight; D009000:Monocytes / Q000737:chemistry*
D003470:Culture Media; D016207:Cytokines; D007375:Interleukin-1