The most commonly used vectors for heterologous protein expression inPichia pastoriscarry its wild-typeHIS4gene and the bacterial ampicillin resistance gene as selectable markers (1 –6 ). TheHIS4gene is relatively large (3 kb), with ill-defined functional boundaries, and its use limits the vectors to selection inhis4auxotrophic strains.HIS4-based vectors are large, typically between 7 and 10 kb in size, creating difficulties for routine cloning manipulations, especially when generating constructions with large or multiple insertions. TheEscherichia colikanamycin gene (from Tn 903) present on someP. pastorisexpression vectors functions as a second marker on someHIS4-based vectors to identify multicopy (i.e., G418 hyperresistant) recombinant strains. However, it cannot be used efficiently as a direct selectable marker for transformation ofP. pastoristo G418 resistance, and its presence leads to a further increase in vector size. The combination of theHIS4, ampicillin, and kanamycin genes adds up to 5.5 kb of “space” in a typical expression vector.
