Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast Pichia pastoris

Human granulocyte colony stimulating factor (hG-CSF) was expressed in the m ethylotrophic yeast Pichia pastoris, using two different constructs which r esulted in proteins with different N-terminal sequences. In the first const ruct, a hexa-histidine tag and enterokinase cleavage site were added to the N-terminus of the protein to achieve one-step separation and exact process ing. In the second construct, the gene was fused to the alpha -MF prepro le ader at the Lys-Arg processing site (without Glu-Ala spacer). ne PCR produc ts were cloned in pPIC9 commercial vector and integrated into the alcohol o xidase region of the host genome. Transformation was done by electroporatio n or spheroplasting. Selection of good producing clones was performed by im munoblot analyses of the supernatants from shake-flask fermentation. Proper processing of the products was confirmed by N-terminal sequencing of the s ecreted proteins. With both plasmid constructs, the target proteins, bearin g the histidine tag or not, represented majority of the secreted proteins. Although the proteins were present in the soluble form, they were highly ag gregated, which interfered with purification. The most efficient way to obt ain monomeric, biologically active protein was complete denaturation by gua nidine-HCl or urea and subsequent renaturation during gel filtration chroma tography.