Anti-6xHIS Tag polyclonal antibody [FITC]

Branched-chain polyamines (BCPAs) are unique polycations found in (hyper)thermophiles. Thermococcus kodakarensis grows optimally at 85 degrees C and produces the BCPA N-4-bis(aminopropyl)spermidine by sequential addition of decarboxylated S-adenosylmethionine (dcSAM) aminopropyl groups to spermidine (SPD) by BCPA synthase A (BpsA). The T. kodakarensis bpsA deletion mutant (DBP1) did not grow at temperatures at or above 93 degrees C, and grew at 90 degrees C only after a long lag period following accumulation of excess cytoplasmic SPD. This suggests that BCPA plays an essential role in cell growth at higher temperatures and raises the possibility that BCPA is involved in controlling gene expression. To examine the effects of BCPA on transcription, the RNA polymerase (RNAP) core fraction was extracted from another bpsA deletion mutant, DBP4 (RNAP(DBP4)), which carried a His-tagged rpoL, and its enzymatic properties were compared with those of RNAP from wild-type (WT) cells (RNAP(WT)). LC-MS analysis revealed that nine ribosomal proteins were detected from RNAP(WT) but only one form RNAP(DBP4). These results suggest that BCPA increases the linkage between RNAP and ribosomes to achieve efficient coupling of transcription and translation. Both RNAPs exhibited highest transcription activity in vitro at 80 degrees C, but the specific activity of RNAP(DBP4) was lower than that of RNAP(WT). Upon addition of SPD and BCPA, both increased the transcriptional activity of RNAP(DBP4); however, elevation by BCPA was achieved at a tenfold lower concentration. Addition of BCPA also protected RNAP(DBP4) against thermal inactivation at 90 degrees C. These results suggest that BCPA increases transcriptional activity in T. kodakarensis by stabilizing the RNAP complex at high temperatures.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases of swine that has adverse effects on the pig industry as a result of direct and indirect loss (Li et al. 2007). Many methods have been applied to control this important viral disease in swine farms, however outbreaks are still taken place. The use of recombinant chimeric swine PKR-Apaf-1 protein (rcPAP) generated from human Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO) concept is an alternative way to control swine viruses. This rcPAP consisted of the Human Immunodeficiency Virus Trans-activator transcription (HIV-TAT) domain, the dsRNA-binding domain of porcine Protein kinase R (PKR) gene and the caspase recruitment domain (CARD) of the porcine Apoptotic Protease-Activating Factor-1 (Apaf-1) gene. This study aimed to produce rcPAP using a bacterial expression system and investigate its biological activity. Recombinant vectors pET-P-A and pQE32-P-A were constructed. The His-tag fusion proteins were expressed in E. coli and purified under native condition with the HiTrap chelating affinity column. The soluble rcPAP produced from the pET-P-A plasmid with a yield of 12.32 mg per liter of bacterial culture media at 25 degrees C for 18 h were 2.4-fold higher than the pQE32-P-A plasmid and were selected for in vitro study. The purified protein reacted with the mouse anti-rcPAP polyclonal antibodies. The rcPAP (80 mu g/mL) induced apoptosis in PRRS virusinfected MARC-145 cells at 48 hpi by increasing the monkey active caspase-3 value by 17.9 fold was higher (9.150 +/- 0.008 vs 0.510 +/- 0.003 ng/mg protein) when compared to uninfected control. The bioactivity in vitro indicates this established protein as a prospective molecule for research to control of PRRS virus infection.