Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

An allergenic 2S storage protein from ricinus communis seeds which is a part of the 2s albumin precursor predicted by C-DNA data

A 1.9s albumin having allergenic activity and denoted Ric c III was isolated from an alcohol extract of defatted Ricinus communis seeds CB-1A, as a homogeneous protein by ion -exchange chromatography on SP-Sephadex, gel filtration on Sephadex G-75 and preparative polyacrylamide gel electrophoresis (6 mg Ric cIII/g CB-1A). The protein contained approximately 98 amino acid residues distributed in 2 chains of 67 anmd 34 residues, a molecular weight of 11.239 based on amino acid composition and pI=4.9 Ric c III can be aligned, on the basis of amino acid composition and partial amino acid sequence data, with residues 18 to 50 (51) and 66 to 130 of the 2S albumin precursor predicted by the cDNA data of S. D. Irwin, J. N. Ken, J. B. C. Findlary and J. M. Lord (Molecular and General Genetics, 222:400-408, 1990). The present data identify Ric c III as the second allergenic 2S storage albumin coded by this DNA

Isolation and partial characterization of antigen 5.1 from the seeds of Ricinus communis, L. (var. amarelo de irece)

Antigen 5.1 was isolated from the most acidic fraction of castor bean allergens (CB-1A) by gel filtration on Sephadex G-75 followed by polyacrylamide gel electrophoresis (PAGE) (yield: 6.2 mg antigen 5.1/g CB-1A). This antigen was homogeneous by the criteria of PAGE, isoelectric focusing, SDS-PAGE, immunoelectrophoresis and gel filtration. The antigen has an apparent molecular mass of 12 ñ 2 kDa and an isoelectric point of pH 5.1. Antigen 5.1 lacks proline, phenylalanine, threonine and tryptophan. It was immunochemically identical to one of the three immunoprecipitation lines presented by CB-1A by the Ouchterlony technique, and was positive when tested (250 *g) by IgE-mediated passive cutaneous anaphylaxis in LOU.M rats

Structural studies on allergen RC-13 from Ricinus communis L.: isolation and characterization of a major glycopeptide

A glycoprotein, RC-13, isolated from Ricinus communis seeds was reduced, S-alkylated and cleaved by trypsin. The tryptic digest was fractionated by ion-exchange chromatography and a glycopeptide was isolated and purified by high-voltage paper electrophoresis. When submitted to amino acid and carbohydrate analyses this major glycopeptide showed the following chemical composition: Lys1, Asp1, Thr2, Ser4, Glu1, Pro2, Gly2, Ala2, Val2, GlcN6, Man6 and Gal8. Hydrazynolysis positioned Ser as the C-terminal residue. It is postulated that this glycopeptide belongs to the C-terminal region of the allergen