Characterization and isolation of an intracellular D-mannose-specific receptor from human promyelocytic HL60 cells.

International audienceMost mammalian macrophages express D-mannose-specific receptor (membrane lectin, Mr 175,000) allowing endocytosis of their ligands, but cells of the monocytic lineage (HL60, U937, monocyte) lack this receptor. However, after permeabilization, promyelocytic, promonocytic cells and monocytes bound fluoresceinylated D-mannose-terminated neoglycoproteins as evidenced by flow cytometry. Under these conditions, confocal analysis confirmed the intracellular membrane localization of the labeling and the absence of nuclear binding. An intracellular D-mannose-specific receptor was isolated from the human promyelocytic cell line HL60, by affinity chromatography on 4-isothiocyanatophenyl alpha-D-mannopyranoside-substituted Affi-gel as a 60,000-Mr membrane protein requiring divalent cations for the ligand binding. Under the same conditions, mouse macrophages were shown to express a 175,000-Mr D-mannose-specific receptor but not the 60,000-Mr receptor

Monofunctional chorismate mutase from Bacillus subtilis: purification of the protein, molecular cloning of the gene, and overexpression of the gene product in Escherichia coli

The monofunctional chorismate mutase from Bacillus subtilis has been purified 2200-fold to homogeneity. The enzyme is a homodimer of subunit Mr = 14,500 and is the smallest natural chorismate mutase that has been characterized. The purified enzyme follows Michaelis-Menten kinetics with a Km of 100 microM and a kcat of 50 s-1, carries no other associated enzymic activities, and is unaffected by any of the aromatic amino acids. The N-terminal amino acid sequence of the protein has been determined, and this information has been used to construct a precise oligonucleotide probe for the gene by means of in vitro DNA amplification from total chromosomal DNA by the polymerase chain reaction. The cloned aroH gene encodes a protein of 127 amino acid residues and is expressed in Escherichia coli. The cloned gene product is indistinguishable from that purified from Bacillus. The aroH coding region was directly subcloned into a phagemid expression vector by means of the polymerase chain reaction. The resulting construct, with the aroH gene positioned behind efficient transcription and translation initiation sequences of E. coli, results in the production of the monofunctional mutase at levels of 30-35% of the soluble cell protein in E. coli transformants. Chorismate mutases comprise a set of functionally related proteins that show little sequence similarity to each other. This diversity stands in contrast to other chorismate-utilizing enzymes

A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest

The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD+ and NADP+, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at ≤180 g MAb/L of resin with the PA eluate, reduced BSA (1-2 log), host cell protein (HCP; 2-3 log), MAb oligomer (31-85%), fragment (from ∼0.8% to <0.1%), and other undesired MAb species. Purity, as measured by non-reducing (NR) SDS-PAGE, was improved 33-85%, to 92-99.5% overall (>99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was ≤1 ng/mg in MAb preparations purified through the three column process and then concentrated and buffer exchanged into the appropriate buffer using tangential flow filtration (TFF). © 2006 Elsevier B.V. All rights reserved