Secretory production of human granulocyte colony-stimulating factor in Escherichia coli.
Human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein, consisting of 174 amino acids, which plays an important role in hematopoietic cell proliferation, differentiation of hemopoietic precursor cells, and activation of mature neutrophilic granulocytes. In this study, secretory production of hG-CSF in the periplasmic space of Escherichia coli using the Bacillus sp. endoxylanase signal peptide was examined. For the efficient expression of hG-CSF gene, the first five codons at the N-terminal were altered based on the E. coli high-frequency codon database. The hG-CSF gene fused to the endoxylanase signal sequence was expressed using an inducible trc promoter. However, recombinant E. coli cells were completely lysed after induction with 1 mM isopropyl-beta-D-thiogalactopyranoside. Insertion of a small oligopeptide (13 amino acids) containing the histidine hexamer and factor Xa cleavage site between the signal peptide and the mature hG-CSF protein allowed successful secretion of hG-CSF into the periplasm without cell lysis. Among the several E. coli strains examined, E. coli BL21(DE3) and E. coli MC4100 allowed production of hG-CSF to the highest levels (20-22% of total proteins) with the secretion efficiencies greater than 98%. The circular dichroism spectra showed that the conformation of purified hG-CSF is almost identical to native hG-CSF.
Jeong
K J
KJ
Metabolic and Biomolecualr Engineering National Research Laboratory, Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea.
Lee
S Y
SY
D001407:Bacillus; D003001:Cloning, Molecular / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases; D004926:Escherichia coli / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D010957:Plasmids; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014995:Xylosidases / Q000235:genetics
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases