Isolation and Purification of Protein Granules from Escherichia coli Cells Overproducing Bovine Growth Hormone

The purification of eukaryotic polypeptides synthesized in Escherichia coli

“…In the phase contrast microscope, inclusion bodies are seen to be highly refractile, while transmission electron microscopy reveals them as amorphous aggregates not enclosed or in contact with a distinct membrane (Schoemaker et al, 1985;Schoner et al, 1985). Electron micrographs show isolated inclusions as spherical in shape ( Fig.…”

“…(a) Transmission electron micrograph of water-washed isolated inclusion bodies in suspension. reported to sediment recombinant inclusion bodies (Olsen, 1985) but values of 5000-12000 g are more generally used George et al, 1985;Schoner et al, 1985). Under these conditions, the inclusion bodies sediment more rapidly than the bulk of the cell debris and purification is achieved.…”

“…3). Detergent or urea (Koths et al, 1985;Schoner et al, 1985) can be used to solubilize the microbial proteins preferentially, leaving the recombinant proteins between 30% and 90% pure. The usefulness of this procedure is underlined by the fact that techniques have been developed to enhance aggregation.…”

Increased production of low molecular weight recombinant proteins in Escherichia coli

“…For HPLC analysis, granules were prepared from 100 mL cultures as described previously (Schoner et al, 1985) and then converted to the S-sulfonate by treating with 1% NaDodS04, 90 mM Na2S03, 12.5 mM Na2S406 (pH adjusted to 8.5 with 10 mM ethylene diamine, which also served as a scavenger for amine specific reaction) at room temperature for 3 h. An aliquot of this reaction mixture was analyzed by reverse-phase HPLC on a 4.5 X 150 mm IBM C8 column with an acetonitrile gradient (20-30%) in 0.1 M ammonium phosphate (pH 7.0) and expression levels were quantified by calibration of the peak area representing S-sulfonate derivative of Met-X,,-IGF-1.…”

Cloning, Purification, and Polymerization ofCapsicum annuumRecombinant α and β Tubulin

“…31) In this study, different concentrations of IPTG (0.1 to 1.0 mM) were compared for their effectiveness in inducing maximal expression of = tubulin. The optimal concentration was 0.2 mM (data not shown).…”