New method for the selection of multicopy transformants of Pichia pastoris, using 3-amino-1,2,4 triazol

The methylotrophic yeast Pichia pastoris has been succcessfully used for th e expression of many heterologous proteins. The level of expression of some of these proteins depends on the copy number of the gene inserted into the yeast genome. Several methods have been reported in the past few years for the isolation of multicopy transformants. One of these methods used an exp ression vector that contains the bacterial kanamycin-resistance gene Tn903k an(r), which confers resistance ro G418. Here, we report different selectio n method in a mutant strain of P. pastoris (his3(-)) based on the resistanc e to 3-amino-1,2,4 triazol, with a vector containing the HIS3 gene from Sac charomyces cerevisiae. Using this selection method we isolated here P. past oris transformants containing several copies of the dextranase gene (dex) f rom Penicillium minioluteum.