Binding properties and solubility of single-chain T cell receptors expressed in E. coli.
The diversity and domain structure of alpha beta T cell receptors (TCR) are similar to immunoglobulins based on sequence homologies, but the three-dimensional structure of the alpha beta-heterodimer has not been solved. To begin structure/function studies, we have compared the properties of a soluble single-chain V alpha V beta TCR (scTCR) expressed in three E. coli systems. The V alpha and V beta regions were expressed with pelB or ompA signal sequences or as a thioredoxin fusion protein. The scTCRs were detected only in the insoluble fraction of the cells and could be solubilized in guanidine and renatured to obtain properly folded scTCR from each system. Only a small fraction (1-5%) of the ompA and pelB scTCRs folded properly. In contrast, the thioredoxin fusion protein exhibited high total yields and a solubility that was ten times higher than the other scTCRs. The thioredoxin fusion protein also bound specifically to the peptide/MHC ligand with a KD of approximately 0.7 microM, as shown by a competitive inhibition assay with Fab fragments that recognize the MHC complex. Furthermore, estimates from saturation binding with antibodies that react with the native TCR indicated that up to 80% of the thioredoxin fusion protein was in the properly folded form. The improved yield, solubility, and binding activity of the thioredoxin-scTCR should make it useful for various structure/ function studies.
Schodin
B A
BA
Department of Biochemistry, University of Illinois, Urbana 61801, USA.
Schlueter
C J
CJ
Kranz
D M
DM
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000276:immunology; D006183:H-2 Antigens / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D012995:Solubility; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic / Q000276:immunology
D006183:H-2 Antigens; D010455:Peptides; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins