title: Optimising the recovery of recombinant thermostable proteins expressed in mesophilic hosts.
abstract: The purification of a thermostable Caldocellum saccharolyticum beta-glucosidase expressed in Escherichia coli was investigated using heat precipitation of unclarified cell homogenates. Heat treatment at 70 degrees C was capable of purification with respect to cell debris, small particulates and the majority of cell protein, although E. coli proteins were even more efficiently removed at 80 degrees C and above. For thermostable proteins expressed in E. coli, a precipitation temperature of 80 degrees C or greater is recommended for optimal removal of contaminant proteins. In small-scale heating trials, heating rate was found to influence enzyme yield significantly. Losses were minimised when 'flash-heating' was employed. The successful single-step removal of particulates, labile protein and nucleic acids was achieved by simultaneous heat-treatment and polyethyleneimine addition, although the purification achieved was additive rather than synergistic.
authors.affiliation: Department of Biochemistry and Molecular Biology, University College London, UK.
authors.forename: N
authors.initials: N
authors.lastname: Kirk
authors.forename: D
authors.initials: D
authors.lastname: Cowan
mesh_terms: D001421:Bacteria, Anaerobic / Q000737:chemistry / Q000378:metabolism*; D011232:Chemical Precipitation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D011094:Polyethyleneimine / Q000737:chemistry; D015202:Protein Engineering; D012313:RNA / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D012313:RNA; D011094:Polyethyleneimine; D001617:beta-Glucosidase