Human G-CSF synthesis using stress-responsive bacterial proteins.
We previously reported that under the stress condition caused by the addition of 2-hydroxyethyl disulfide, a thiol-specific oxidant, to growing cultures of Escherichia coli BL21(DE3), a population of stress-responsive proteins [peptidyl-prolyl cis-trans isomerase B (PpiB), bacterioferritin (Bfr), putative HTH-type transcriptional regulator yjdC (YjdC), dihydrofolate reductase (FolA), chemotaxis protein cheZ (CheZ), and glutathione synthetase (GshB)] were significantly upregulated when compared with the nonstress condition. When those stress-responsive proteins were used as fusion partners for the expression of human granulocyte colony-stimulating factor (hG-CSF), the solubility of hG-CSF was dramatically enhanced in E. coli cytoplasm, whereas almost all of the directly expressed hG-CSF were aggregated to inclusion bodies. In addition, the spectra of circular dichroism measured with the purified hG-CSF were identical to that of standard hG-CSF, implying that the synthesized hG-CSF has native conformation. These results indicate that the bacterial stress-responsive proteins could be potent fusion expression partners for aggregation-prone heterologous proteins in E. coli cytoplasm.
Song
Jong-Am
JA
Department of Chemical and Biological Engineering, Korea University, Seoul, South Korea.
Han
Kyung-Yeon
KY
Park
Jin-Seung
JS
Seo
Hyuk-Seong
HS
Ahn
Keum-Young
KY
Lee
Jeewon
J
D002942:Circular Dichroism / Q000379:methods; D004220:Disulfides / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects*; D029968:Escherichia coli Proteins / Q000096:biosynthesis*; D000431:Ethanol / Q000031:analogs & derivatives* / Q000494:pharmacology; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D016877:Oxidants / Q000494:pharmacology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012995:Solubility
D004220:Disulfides; D029968:Escherichia coli Proteins; D016877:Oxidants; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor; D000431:Ethanol; C031319:2-hydroxyethyl disulfide