High-level production of a secreted, heterodimeric alpha beta murine T-cell receptor in Escherichia coli.
J Immunol Methods
; 206(1-2): 163-9, 1997 Aug 07.
Article
em En
| MEDLINE
| ID: mdl-9328579
ABSTRACT
For structural studies, high-level production of properly folded, disulfide-linked, unglycosylated protein in E. coli is an attractive alternative to production in eukaryotic systems. We describe here the production of heterodimeric, murine D10 T-cell receptor (sD10TCR) in E. coli as a secreted leucine zipper (LZ) fusion protein. Two genes, one (alpha-acid) encoding the alpha-chain variable and constant domains (V alpha and C alpha) of D10 TCR fused to an LZ 'acid' encoding sequence and the other (beta-base) encoding the beta-chain variable and constant domains (V beta and C beta) fused to an LZ 'base' encoding sequence, were co-expressed from a bacteriophage T7 promoter as a dicistronic message. Secreted alpha-acid and beta-base proteins formed proper inter- and intra-chain disulfide bonds in the periplasm, bypassing the need for in vitro protein refolding. Complementary LZ sequences facilitated the formation of alpha beta heterodimers. sD10TCR-LZ was purified by affinity chromotography using a D10 TCR clonotype-specific monoclonal antibody (mAb 3D3). Typical yields of purified protein were 4-5 mg/l of culture. Purified sD10TCR-LZ was reactive with a panel of conformationally sensitive TCR-specific monoclonal antibodies, consistent with its conformational integrity and appeared to be suitable for structural studies by X-ray crystallography or NMR spectroscopy.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes de Fusão
/
Receptores de Antígenos de Linfócitos T alfa-beta
/
Escherichia coli
Limite:
Animals
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Estados Unidos