High-level production of a secreted, heterodimeric alpha beta murine T-cell receptor in Escherichia coli.
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.
Golden
A
A
Procept Inc., Cambridge, MA 02139, USA.
Khandekar
S S
SS
Osburne
M S
MS
Kawasaki
E
E
Reinherz
E L
EL
Grossman
T H
TH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D016350:Leucine Zippers / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D000911:Antibodies, Monoclonal; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins