Clinical grade production and characterization of a fusion protein comprised of the chemokine CCL2-ligand genetically fused to a mutated and truncated form of the Shiga A1 subunit
| Autor: | Bonny S.F. Choy, Liliana Perdomo, Laura M. McIntosh, Barbara K. Finck, John R. McDonald, Hongsheng Su, Michael Jack |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
CCR2
Cell Survival Protein subunit Recombinant Fusion Proteins Population Molecular Sequence Data Biology Inclusion bodies Shiga Toxin Reticulocyte Protein A/G medicine Escherichia coli Humans Amino Acid Sequence education Chemokine CCL2 Cell Proliferation Protein Synthesis Inhibitors education.field_of_study Chromatography Adenine Ligand (biochemistry) Molecular biology Fusion protein medicine.anatomical_structure Biochemistry biology.protein Ribosomes Biotechnology Protein Binding |
| Zdroj: | Protein expression and purification. 66(2) |
| ISSN: | 1096-0279 |
| Popis: | First generation chemokine ligand-Shiga A1 (SA1) fusion proteins (leukocyte population modulators, LPMs) were previously only obtained in small quantities due to the ribosomal inactivating protein properties of the SA1 moiety which inhibits protein synthesis in host cells. We therefore employed 4-aminopyrazolo[3,4-d]-pyrimidine, an inhibitor of Shiga A1, to allow the growth of these cells prior to induction and during the expression phase post-induction with IPTG. Scale-up allowed the production of gram quantities of clinical grade material of the lead candidate, OPL-CCL2-LPM. A manufacturing cell bank was established and used to produce OPL-CCL2-LPM in a fed-batch fermentation process. Induction of the expression of OPL-CCL2-LPM led to the production of 22.47 mg/L per OD(600) unit. The LPM was purified from inclusion bodies using solubilization, renaturation, refolding and chromatography steps. The identity and purity of the OPL-CCL2-LPM was determined using several analytical techniques. The product retained the ability of the SA1 moiety to inhibit protein synthesis as measured in a rabbit reticulocyte lysate cell-free protein synthesis assay and was cytotoxic to target cells. Binding studies established that the protein exerts its effects via CCR2, the cognate receptor for CCL2. Clinical trials in inflammatory nephropathies are planned. |
| Databáze: | OpenAIRE |
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