title: Isolation of carcinoembryonic antigen N-terminal domains (N-A1) from soluble aggregates.
abstract: Carcinoembryonic antigen (CEA) was identified as a prominent tumor-associated antigen in human colorectal cancer and it is still intensively investigated. However, its physiological role remains unclear. The CEA molecule is composed of seven highly hydrophobic, immunoglobulin-like domains, six of which contain a single disulphide bridge. The production of recombinant protein containing Ig-like domains in bacterial expression systems often results in partial degradation or insolubility due to aggregation hampering the analysis of their native structure and function. Here, we present a new method of expression and purification of CEA N-terminal domains (N-A1) fused to MBP in Escherichia coli. In order to optimize the expression and purification of CEA N-A1 domains we evaluated bacteria cultivation conditions, the length of N-A1 domains, fusion systems (GST- and MBP-tag), IPTG concentrations and protein purification conditions. We have found that MBP-N-A1 fusion protein digested with TEV protease forms soluble aggregates composed of N-A1 domains and incompletely digested MBP-N-A1 fusion protein. Using 1.25 M guanidinium chloride (GdmCl) as a component of the elution buffer we were able to achieve an almost complete dissociation of the aggregates. The dissociation was monitored by circular dichroism and fluorescence measurements. The CD spectra and Ellman's assay suggest that the conformation of N-A1 domains and their disulphide bonds are correct.
authors: Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, PL-53-114 Wroclaw, Poland.
authors: Hanna
authors: H
authors: Czepczyńska-Krężel
authors: Marcin
authors: M
authors: Czerwinski
authors: Artur
authors: A
authors: Krężel
authors: Anna
authors: A
authors: Krop-Watorek
mesh_terms: D002272:Carcinoembryonic Antigen / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019791:Guanidine; D006801:Humans; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility
chemical_list: D002272:Carcinoembryonic Antigen; D004220:Disulfides; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D019791:Guanidine