A method for rapid screening of recombinant proteins for recognition by T lymphocytes.
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.
Hickling
J K
JK
ImmuLogic Pharmaceutical Corporation, Palo Alto, CA 94304.
Jones
K R
KR
Yuan
B
B
Rothbard
J B
JB
Bülow
R
R
D000818:Animals; D001483:Base Sequence; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*
D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins