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525307 
Journal Article 
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 
Su, HS; Jack, M; McIntosh, LM; Perdomo, L; Choy, BSF; Finck, BK; McDonald, JR 
2009 
1 
Protein Expression and Purification
ISSN: 1046-5928 
66 
2 
149-157 
English 
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 OD600 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. (C) 2009 Elsevier Inc. All rights reserved. 
Chemokine; CCL2; Fusion protein; Shiga A1 subunit; GMP; monocyte chemoattractant protein-1; ribosome-inactivating proteins; chemotactic protein-1; receptor nomenclature; international union; mcp-1 receptor; macrophages; identification; pharmacology; inhibition