title: Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2.
abstract: The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N-terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.
authors.affiliation: Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
authors.forename: E A
authors.initials: EA
authors.lastname: Barron-Casella
authors.forename: T S
authors.initials: TS
authors.lastname: Kickler
authors.forename: O C
authors.initials: OC
authors.lastname: Rogers
authors.forename: J F
authors.initials: JF
authors.lastname: Casella
mesh_terms: D000483:Alleles; D000906:Antibodies / Q000302:isolation & purification; D001327:Autoimmune Diseases / Q000097:blood; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000096:biosynthesis / Q000302:isolation & purification; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics; D011693:Purpura / Q000097:blood / Q000209:etiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013921:Thrombocytopenia / Q000097:blood / Q000276:immunology; D065227:Transfusion Reaction
chemical_list: D000906:Antibodies; D017931:DNA Primers; D000939:Epitopes; D010980:Platelet Membrane Glycoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase