Purification and characterization of human interleukin‐1β expressed in recombinant Escherichia coli

Abstract

The high-level expression of human interleukin-la in Escherichiu coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion-and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N-and C-terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vifro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1 /LAF) assay. The specific activities determined with the IL-1/MCF and IL-l/ LAF assays, are 2 x lo7 and 4 x lo7 units mg-', respectively.The term interleukin-1 (IL-1) applies to at least two cytokines which play a role in immunity and in the pathophysiology of inflammation [I -31. Preparations of authentic IL-1 tend to be contaminated with other cytokine mediators which can complicate the interpretation of sensitive IL-1 bioassays (see, for example [4]). The availability of IL-1 produced by recombinant DNA technology would eliminate the problem of potential contamination and provide a reliable source of well characterized cytokine.Recently, the coding sequences for human IL-la and have been cloned from peripheral blood monocytes and their expressed products shown to possess IL-1 activities [5, 61. Using published sequence data we have isolated clones of IL-lP cDNA from a library constructed from mRNA expressed in human histiocytic lymphoma U937 cells. Here we report the expression of the mature IL-la protein in Escherichiu coli, a method for its purification, and the physiochemical and biological properties of the purified protein. MATERIALS AND METHODS Bacterial hosts and plusmids

Differential binding of IL‐ 1α and IL‐ 1β to receptors on B and T cells

“…Human recombinant IL-I/b' (Sclavo-De.Bi., Siena and Cassina de' Pecchi, Italy), expressed in E. coli and purified as in [7], was radioiodinated with [~2Sl]di-iodo Bolton-Hunter (4400 Ci/mmol, New England Nuclear, Boston, MA) following the manufacturer's instructions. The specific activities obtained were routinely around 1.2 x 106 cpm/pmol for IL-l~'and 5.4 x 106 cpm/pmol for IL-lo<.…”

N‐terminal‐methionylated interleukin‐1β has reduced receptor‐binding affinity

“…In the case of IL-2, however, Yamada et al [5] have reported that there is no difference in biological activity between N-terminal-methionylated and non-methionylated protein. In this context, Wingfield et al [1] also saw little difference in biological activity between methionylated and non-methionylated IL-1/?. (Although the methionylated IL-I~ was consistently at least 2-fold less active than the non-methionylated protein, this was not considered significant in view of the inherent variability in the assay used.)…”

“…Isoelectric focusing on thin-layer polyacrylamide gels and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were performed according to [1,2]. ment of the non-methionylated IL-IA?…”

“…The purification and characterization of recombinant-derived interleukin-lz] (IL-ld) have been recently described [1,2]. The purified protein was found to be a mixture of 80%o correctly processed protein (N-terminus = Ala) and 20% of an N-terminal-methionylated form.…”

Preparation, characterization and application of interleukin‐1β mutant proteins with surface‐accessible cysteine residues

“…The mutant proteins, K27C and K138C, were constructed such that the lysines at positions27 and 138, respectively, were replaced by cysteine residues. Fermentation and proteinpurification procedures were essentially as described previously for the recombinant-derived wild-type protein [6]. The method used for purification of the mutant proteins differed from that used for the wild-type protein as follows.…”