Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
1-2
|
| pubmed:dateCreated |
1997-10-23
|
| pubmed:databankReference | |
| pubmed:abstractText |
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.
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
Aug
|
| pubmed:issn |
0022-1759
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
7
|
| pubmed:volume |
206
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
163-9
|
| pubmed:dateRevised |
2003-11-14
|
| pubmed:meshHeading |
pubmed-meshheading:9328579-Amino Acid Sequence,
pubmed-meshheading:9328579-Animals,
pubmed-meshheading:9328579-Antibodies, Monoclonal,
pubmed-meshheading:9328579-Antigen-Antibody Reactions,
pubmed-meshheading:9328579-Dimerization,
pubmed-meshheading:9328579-Escherichia coli,
pubmed-meshheading:9328579-Leucine Zippers,
pubmed-meshheading:9328579-Mice,
pubmed-meshheading:9328579-Molecular Sequence Data,
pubmed-meshheading:9328579-Receptors, Antigen, T-Cell, alpha-beta,
pubmed-meshheading:9328579-Recombinant Fusion Proteins,
pubmed-meshheading:9328579-Solubility
|
| pubmed:year |
1997
|
| pubmed:articleTitle |
High-level production of a secreted, heterodimeric alpha beta murine T-cell receptor in Escherichia coli.
|
| pubmed:affiliation |
Procept Inc., Cambridge, MA 02139, USA.
|
| pubmed:publicationType |
Journal Article
|