ABSTRACT
CD28 and CTLA-4 are homologous cell surface proteins expressed by T cells. CD28 is constitutively expressed by most T cells, whereas CTLA-4 is expressed by activated T cells. Both proteins are ligands for the costimulatory molecules CD80 and CD86 expressed by activated B cells, macrophages, and dendritic cells. A fusion protein comprising the CTLA-4 extracellular domain joined to a human immunoglobulin heavy chain constant region (CTLA4Ig) binds CD80 and CD-86 with high affinity and inhibits CD80/CD86-dependent immune responses in vitro and in vivo. Attempts at producing the CTLA-4 extracellular domain as an unfused protein have met with limited success. Here we describe the expression and purification of the CTLA-4 extracellular domain as a nonfused protein in Escherichia coli. The 12.5-kDa CTLA-4 extracellular domain was insoluble when expressed in E. coli and required denaturation, reduction, and refolding steps to become soluble and assume its proper conformation. The protein refolded into a mixture of monomers, disulfide-linked dimers, and higher order disulfide-linked aggregates. sCTLA-4 dimers were the predominant refold form when air was used as the oxidizing agent during the refold procedure. Purified sCTLA-4 dimers were 10- to 50-fold more potent than sCTLA-4 monomers at inhibiting T cell activation using a CD80-dependent in vitro bioassay.
Subject(s)
Antigens, Differentiation/chemistry , Antigens, Differentiation/genetics , Immunoconjugates , Abatacept , Animals , Antigens, CD , Antigens, Differentiation/metabolism , B7-1 Antigen/genetics , B7-1 Antigen/metabolism , Base Sequence , CHO Cells , CTLA-4 Antigen , Cell Line , Cricetinae , DNA Primers/genetics , Dimerization , Escherichia coli/genetics , Genetic Vectors , Humans , In Vitro Techniques , Jurkat Cells , Lymphocyte Activation , Plasmids/genetics , Protein Folding , Protein Structure, Quaternary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Solubility , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Two soluble receptors of tumour necrosis factor were evaluated for development as potential therapeutic agents for inflammatory disease. The recombinant human soluble Type I and Type II TNF receptors, rsTNF-RI and rsTNF-RII, were expressed at high levels in E. coli, refolded, and chromatographically purified to homogeneity. The potencies of both recombinant soluble receptors were similar to their naturally occurring soluble receptors. In in vitro cytotoxicity and competitive binding assays, both recombinant soluble receptors functioned to inhibit the biological effects of rhTNF-alpha although rsTNF-RI was a 5 to 30 fold more potent inhibitor of rhTNF-alpha than was rsTNF-RII or a truncated form of the soluble receptor, TNF-RII delta. In in vivo experiments in mice, rsTNF-RI was a better inhibitor than rsTNF-RII delta of rhTNF-alpha-stimulated changes in the percentages of circulating lymphocytes and neutrophils, influx of neutrophils into the peritoneal cavity, and serum IL-6 induction. At molar ratios of 0.1:1 and 0.01:1 (rsTNF-R:rhTNF-alpha), using the rsTNF-I or rsTNF-II delta, there was a trend towards enhancement of the induction of IL-6. However, higher ratios of either rsTNF-RI or rsTNF-RII delta significantly inhibited the rhTNF-alpha-stimulated increase in serum IL-6 levels. In a murine model of cytokine-induced shock, either rsTNF-RI or rsTNF-RII delta provided protection against the lethality of shock induced by a synergistic combination of rhTNF-alpha and rhIL-1 beta. Based on the results of these experiments, the rsTNF-RI was chosen as the better candidate for development as an anti-inflammatory agent.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Receptors, Tumor Necrosis Factor/physiology , Recombinant Fusion Proteins/pharmacology , Tumor Necrosis Factor-alpha/physiology , Animals , Ascitic Fluid/cytology , Base Sequence , Binding, Competitive , Cloning, Molecular , Drug Synergism , Female , Humans , Interleukin-1/toxicity , Interleukin-6/blood , Interleukin-6/metabolism , L Cells/drug effects , Leukocyte Count/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , Neoplasm Proteins/pharmacology , Peptide Fragments/pharmacology , Receptors, Tumor Necrosis Factor/classification , Recombinant Fusion Proteins/biosynthesis , Shock/chemically induced , Shock/prevention & control , Solubility , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/pharmacology , Tumor Necrosis Factor-alpha/toxicitySubject(s)
Coliphages , Genes , Mutation , Adsorption , Autoradiography , Carbon Radioisotopes , Chromosome Mapping , Coliphages/drug effects , Coliphages/growth & development , DNA Viruses/drug effects , DNA Viruses/growth & development , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Microscopy, Electron , Molecular Weight , Phenotype , Polyethylene Glycols/pharmacology , Viral Plaque AssayABSTRACT
Escherichia coli strain 9D3 possesses a highly temperature-sensitive valyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.9). Since 9D3 is a rel(+) strain, it cannot carry out net RNA synthesis at high temperature. A 100-mug amount of chloramphenicol (CAP) per ml added in the absence of valine cannot stimulate RNA synthesis. Either 300 mug of CAP or 100 mug of CAP plus 50 mug of valine per ml, however, promotes nearly maximal RNA synthesis. These results can be understood as follows. (i) Valyl-tRNA is required for net RNA synthesis, (ii) the synthetase lesion is incomplete, (iii) the rate of mutant acylation of tRNA(val) at high temperature is valine-dependent, and (iv) the CAP concentration determines the rate of residual protein synthesis. Data are also presented which demonstrate that the rate of net RNA synthesis can greatly increase long after the addition of CAP, if the amount of valyl-tRNA increases.
