A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments.
AUTOR(ES)
Chang, H C
RESUMO
Generation of soluble T-cell receptor (TCR) molecules by a variety of genetic engineering methods has been hampered by inefficient pairing of alpha and beta subunits in the absence of their respective transmembrane regions and associated CD3 components. To overcome this obstacle, we have added 30-amino acid-long segments to the carboxyl termini of alpha and beta extracellular domains via a cleavable flexible linker. These peptide segments (BASE-p1 for alpha and ACID-p1 for beta) have been previously shown to selectively associate to form a stable heterodimeric coiled coil termed a leucine zipper. Homodimeric structures are not permitted due to electrostatic repulsion among amino acid side chains. Expression of a representative TCR-leucine zipper fusion protein in a baculovirus expression system results in production of alpha beta TCR heterodimer at 0.6-1.4 mg/liter. This yield is 5- to 10-fold greater than that of the TCR expressed in the absence of the synthetic leucine zipper sequence. The structure of the TCR component of the fusion heterodimer was judged to be native when probed with a panel of 17 mAbs specific for alpha and beta constant and variable domains. A mAb specific for the isolated BASE-p1/ACID-p1 coiled coil was also generated and shown to react with the TCR fusion protein. The above technology should be broadly useful in the efficient production and purification of TCRs as well as other heterodimeric proteins.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=45240Documentos Relacionados
- Two distinct alpha beta T-cell lineages can be distinguished by the differential usage of T-cell receptor V beta gene segments.
- Engineered secreted T-cell receptor alpha beta heterodimers.
- Ig alpha and Ig beta are functionally homologous to the signaling proteins of the T-cell receptor.
- Two distinct T-cell receptor alpha-chain transcripts in a rabbit T-cell line: implications for allelic exclusion in T cells.
- Rearrangement and expression of the alpha- and beta-chain genes of the T-cell antigen receptor in functional murine suppressor T-cell clones.