Mammalian Expression Vectors for Epitope Tag Fusion Proteins that Are Toxic in E. coli

Abstract

The amplification from the crude lysates of the recombinant P. pastoris strains KM71 (pPIC9-MYO)-Mut S 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)-Mut S 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.

Association of the Kinesin‐Binding Domain of RanBP2 to KIF5B and KIF5C Determines Mitochondria Localization and Function

“…Deletion mutagenesis was carried out with pairs of primers against domains of interest, described in the figure legends. The PCR products were subcloned into pGEX‐KG (41), pExpress‐EGFP (42) and pDEST‐YFP vectors.…”

Caspase-activated PAK-2 Is Regulated by Subcellular Targeting and Proteasomal Degradation

“…The bicistronic retroviral expression vector pRetroIRES-GFP 2 was generated from RetroTet RTRg(Ϫ)gfp (Dr. Helen M. Blau, Stanford University). The cloning vector pKoz/EGFP and the expression vector pExpress/EGFP were generated as described previously (26,27). pMT107 and pMT123 expression vectors for His-ubiquitin (His-Ub) and HA-ubiquitin (HA-Ub), respectively, were gifts from Dr. D. Bohmann, Rochester University.…”

Identification and Characterization of PS-GAP as a Novel Regulator of Caspase-activated PAK-2

“…Agaroseconjugated anti-HA polyclonal antibody was obtained from Santa Cruz Biotechnology. Mammalian expression vectors pExpress/HA and pRetroIRES/GFP were generated previously (42,45). Mammalian expression clones for FLAG-tagged PAK-2p34 and PAK-2p34-K278R in pRetroIRES/GFP (42) and for FLAG-tagged PAK-2, PAK-2-L106F, and PAK-2-K278R in pRevTRE 2 were generated previously.…”