Mammalian expression vectors for epitope tag fusion proteins that are toxic in E. coli.

lysates of the recombinant P. pastoris strains KM71 (pPIC9-MYO)-MutS and GS115 (pPIC9-MYO)-Mut+, which contained the integration of the myostatin gene to chromosomal DNA, was completed. When Taq DNA polymerase was used for amplification, we observed either no or nonspecific amplification products. The positive control sample yielded the 831-bp specific fragment. When the HOTStar Taq DNA polymerase was used for the amplification, the 831-bp specific amplification product was visible. The wild-type gene AOX1 2.2-kb fragment was co-amplified in GS115-derived strains. The fragment is not visible in KM71-derived strains, because the gene is deleted from the parent strain. The highest yield was attained with 10 min of boiling at 80°C (Figure 1). Changing the volume of amplification mixture did not influence the result. No changes were found when the annealing temperature and number of amplification cycles were changed. Similar results were observed when the recombinant yeast P. pastoris strain KM71 (pPIC9-LEP)-MutS with cloned gene for leptin was amplified using the same procedure. The results are demonstrated in Figure 2. In this experiment, a specific 912-bp product was observed as a positive result. No product from the 2.2-kb wild-type gene fragment was visible in these KM71-derived strains. From the presented results we can deduce that crude yeast lysates contain many PCR inhibitors, which decreases the specificity of the amplification and the amount of amplification product. In contrast to the amplification from the crude lysates of some bacterial cells, only some robust Taq DNA polymerases can overcome these inhibitors. However, fundamentally, the amplification from crude yeast lysates is possible, and the described method can be used as an inexpensive and quick alternative to isolation of chromosomal DNA from yeast cells.