Purification and characterization of recombinant human leukocyte interferon (IFLrA) with monoclonal antibodies.

T Staehelin,D.S Hobbs,H Kung,C.Y Lai,S Pestka

doi:10.1016/s0021-9258(19)68827-7

Abstract

Recombinant human leukocyte interferon produced in bacteria (IFLrA) was purified to homogeneity with the use of monoclonal antibodies against leukocyte interferon. The purified interferon exhibited a single band of Mr = approximately 19,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis and the NH2-terminal sequence were consistent with the sequence predicted from the DNA. Some of the purified product contained NH2-terminal methionine; the terminal methionine was removed from the rest of the chains.

Highlights

Recombinant human leukocyte interferon produced in bacteria (IFLrA) was purified to homogeneity with theuseofmonoclonalantibodies against leukocyte interferon
With the isolation of 13 monoclonal antibodies tohuman leukocyte interferon [3], it was one of our goals to use one or more of them for the purifkationof the interferon produced in bacteria
The major protein contaminating the interferon after the monoclonal antibody column exhibited M, = 45,000 and possiblywas bacterial elongation factor EF-Tu or a dimer of ISLrA (Figs. 3 and 4)

Summary

Purification of recombinant leukocyte interferon

Protein was determined by the method ofLowry et al [6] with crystalline bovine serum albumin as standard. Interferon activity was determined by acytopathic effect inhibition assay with vesicular stomatitis virus and MDBK cells (bovine kidney cellline) as described. Interferon activity was determined by a radioimmunoassay performed with monoclonal antibodies [8] or by a cytopathiceffect inhibition assay with vesicular stomatitis virus and either bovine MDBK cells or human AG-1732 cells as reported [9]. The 1-propanol concentration increased linearly to 20% (v/v) in the fust min, followed by elution of interferon with a linear 20-40%(v/v) gradient over the 3 h. The entire sample was applied to an Ultrasphere-octyl column (4.6 X 250 mm; 5-pm particle size; Altex Scientific) and eluted at 0.5 ml/min by a linear 0-408 (v/v) 1-propanol gradient in 0.1 M formate/0.03 M pyridine for 3 h. The column effluent was monitored with the automatic fluorescamine system [7]

Purification of Recombinant Leukocyte Interferon

RESULTS

DISCUSSION

LY s

Methods