Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli

Luciano G. Frigeri, Michael W. Robertson, Fu-Tong Liu

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

IgE-binding protein (eBP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine β-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (∈BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat ∈BP cDNA, we have succeeded in expressing recombinant ∈BP in Escherichia coli. Milligram quantities of homogeneous ∈BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant ∈BP (r∈BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat ∈BP. The purified r∈BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine > thiodigalactoside > lactose ≫ D-galactose > L-arabinose, an order identical to that exhibited by native ∈BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although ∈BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r∈BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of ∈BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.

Original languageEnglish (US)
Pages (from-to)20763-20769
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number34
StatePublished - Dec 5 1990

ASJC Scopus subject areas

  • Biochemistry

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    Frigeri, L. G., Robertson, M. W., & Liu, F-T. (1990). Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli. Journal of Biological Chemistry, 265(34), 20763-20769.