Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli.
IgE-binding protein (epsilon BP) 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 beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon 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 epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon 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 epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.
Frigeri
L G
LG
Medical Biology Institute, La Jolla, California 92037.
Robertson
M W
MW
Liu
F T
FT
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007073:Immunoglobulin E / Q000378:metabolism*; D015471:Leukemia, Basophilic, Acute / Q000276:immunology; D008222:Lymphokines / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D029584:Prostatic Secretory Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D008222:Lymphokines; D029584:Prostatic Secretory Proteins; D011994:Recombinant Proteins; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors; D007073:Immunoglobulin E