Small Vectors for Expression Based on Dominant Drug Resistance with Direct Multicopy Selection

The most commonly used vectors for heterologous protein expression in Pichia pastoris carry its wild-type HIS4 gene and the bacterial ampicillin resistance gene as selectable markers (1 –6 ). The HIS4 gene is relatively large (3 kb), with ill-defined functional boundaries, and its use limits the vectors to selection in his4 auxotrophic 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. The Escherichia coli kanamycin gene (from Tn 903) present on some P. pastoris expression vectors functions as a second marker on some HIS4 -based vectors to identify multicopy (i.e., G418 hyperresistant) recombinant strains. However, it cannot be used efficiently as a direct selectable marker for transformation of P. pastoris to G418 resistance, and its presence leads to a further increase in vector size. The combination of the HIS4 , ampicillin, and kanamycin genes adds up to 5.5 kb of “space” in a typical expression vector.