Development of the G418 Resistance Gene as a Primary Selectable Marker for Pichia pastoris
Pichia Protein Expression Conference
San Diego, CA
October 8-11, 2006
Date of Presentation
There are relatively few dominant selectable markers available for the transformation of Pichia pastoris. The major markers, the zeocin and blasticidin resistance genes, require expensive antibiotics and extensive screening in order to isolate transformants with high copy number. The G418 resistance gene has been utilized for selection of multicopy strains, but only as a secondary selectable marker after primary selection with a biosynthetic marker such as HIS4. We have modified the G418 resistance gene so that it can now be used in P. pastoris for direct selection. Transformation with this new marker generates colonies of varying size on plates containing the antibiotic. Compared to small colonies, larger colonies harbor a greater number of plasmids containing the G418 resistance gene and express higher levels of a reporter gene that is carried in the vector. Besides adding greater flexibility to the P. pastoris system, this G418 selectable marker is more economical and provides an easier way to identify multicopy strains.
Lin-Cereghino, G. P.,
Development of the G418 Resistance Gene as a Primary Selectable Marker for Pichia pastoris.
Paper presented at Pichia Protein Expression Conference in San Diego, CA.