Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast pichia pastoris.
Human granulocyte colony stimulating factor (hG-CSF) was expressed in the methylotrophic yeast Pichia pastoris, using two different constructs which resulted in proteins with different N-terminal sequences. In the first construct, a hexa-histidine tag and enterokinase cleavage site were added to the N-terminus of the protein to achieve one-step separation and exact processing. In the second construct, the gene was fused to the alpha-MF prepro leader at the Lys-Arg processing site (without Glu-Ala spacer). The PCR products were cloned in pPIC9 commercial vector and integrated into the alcohol oxidase region of the host genome. Transformation was done by electroporation or spheroplasting. Selection of good producing clones was performed by immunoblot analyses of the supernatants from shake-flask fermentation. Proper processing of the products was confirmed by N-terminal sequencing of the secreted proteins. With both plasmid constructs, the target proteins, bearing the histidine tag or not, represented majority of the secreted proteins. Although the proteins were present in the soluble form, they were highly aggregated, which interfered with purification. The most efficient way to obtain monomeric, biologically active protein was complete denaturation by guanidine-HCl or urea and subsequent renaturation during gel filtration chromatography.
Lasnik
M A
MA
LEK dd, R&D, Ljubljana, Slovenia.
Porekar
V G
VG
Stalc
A
A
D001709:Biotechnology / Q000379:methods; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor