Recombinant-DNA-derived bovine growth hormone from Escherichia coli. 1. Demonstration that the hormone is expressed in reduced form, and isolation of the hormone in oxidized, native form.
The isolation of bacterially synthesized, recombinant-DNA-derived, bovine growth hormone (r-bGH) with native structure is described. The r-bGH is found in insoluble form, in a pellet fraction, after cell breakage and centrifugation. Cell envelope components (protein, lipid, endotoxin) and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction. We demonstrate that the r-bGH is largely reduced until solubilized using 6 M guanidine/HCl. Air oxidation is then carried out, in the presence of the guanidine/HCl. The oxidation results in a mixture of about one-third disulfide-linked oligomers and two-thirds oxidized monomer. The latter may include some incorrectly oxidized material, but appears to be mostly correctly oxidized. The oxidized monomer is isolated by gel filtration in the presence of guanidine/HCl. Subsequent guanidine/HCl removal leads to refolded, oxidized r-bGH. All steps in the procedure, in particular the oxidation and refolding steps, can be carried out at relatively high protein concentrations.
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D013006:Growth Hormone / Q000096:biosynthesis* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012995:Solubility
D011994:Recombinant Proteins; D013006:Growth Hormone