A method for rapid screening of recombinant proteins for recognition by T lymphocytes

• Published in: European journal of immunology Vol. 22; no. 8; p. 1983
• Main Authors: Hickling, J K, Jones, K R, Yuan, B, Rothbard, J B, Bülow, R
• Format: Journal Article
• Language: English
• Published: Germany 01.08.1992
• Subjects: Animals; Base Sequence; Epitopes; Hemagglutinin Glycoproteins, Influenza Virus; Hemagglutinins, Viral - immunology; Molecular Sequence Data; Peptide Fragments - immunology; Rats; Recombinant Fusion Proteins - immunology; T-Lymphocytes - immunology
• ISSN: 0014-2980
• DOI: 10.1002/eji.1830220805

A simple, cost-effective method is described that allows rapid screening of recombinant protein sequences for their ability to stimulate T cells. Individual microcultures of E. coli each expressing a gene product or peptide sequence fused to protein A are grown in 96-well plates. Following lysis of the bacteria, the fusion peptide is readily captured with immobilized immunoglobulin in tissue culture wells. No further purification is required. T lymphocytes plus appropriate antigen-presenting cells are added directly to the wells and assayed for proliferation. The DNA in bacteria from wells stimulating T cell proliferation is then sequenced. The technique allows rapid mapping of T cell epitopes by facilitating screening of truncation mutants without extensive purification. Described here is a further application of the technique to study monosubstituted analogues of a known T cell epitope.