title: Optimization of human granulocyte macrophage-colony stimulating factor (hGM-CSF) expression using asparaginase and xylanase gene's signal sequences in Escherichia coli.
abstract: The toxicity of the recombinant protein towards the expression host remains a significant deterrent for bioprocess development. In this study, the expression of human granulocyte macrophage-colony stimulating factor (hGM-CSF), which is known to be toxic to its host, was enhanced many folds using a combination of genetic and bioprocess strategies in Escherichia coli. The N terminus attachment of endoxylanase and asparaginase signal sequences from Bacillus subtilis and E. coli, respectively, in combination with and without His-tag, considerably improved expression levels. Induction and media optimization studies in shake flask cultures resulted in a maximal hGM-CSF concentration of 365 mg/L in the form of inclusion bodies (IBs) with a specific product yield (Y (P/X)) of 120 mg/g dry cell weight in case of the asparaginase signal. Culturing the cells in nutrient rich Terrific broth maintained the specific product yields (Y (P/X)) while a 6.6-fold higher volumetric concentration of both product and biomass was obtained. The purification and refolding steps were optimized resulting in a 95% pure protein with a fairly high refolding yield of 45%. The biological activity of the refolded protein was confirmed by a cell proliferation assay on hGM-CSF dependent human erythroleukemia TF-1 cells. This study demonstrated that this indeed is a viable route for the efficient production of hGM-CSF.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Yogender Pal
authors.initials: YP
authors.lastname: Khasa
authors.forename: Amardeep
authors.initials: A
authors.lastname: Khushoo
authors.forename: Suman
authors.initials: S
authors.lastname: Tapryal
authors.forename: K J
authors.initials: KJ
authors.lastname: Mukherjee
mesh_terms: D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism / Q000473:pathology; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D058849:Protein Refolding; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D006639:Histidine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D043364:Endo-1,4-beta Xylanases; D001215:Asparaginase