Biotinylated-sortase self-cleavage purification (BISOP) method for cell-free produced proteins

<p>Abstract</p> <p>Background</p> <p>Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in <it>Escherichia coli</it>. This method, however, is yet to be applied to the cell-free based protein production.</p> <p>Results</p> <p>The histidine tag-based self-cleavage method for purifying proteins produced by the wheat cell-free protein synthesis system showed high background, low recovery, and unexpected cleavage between the N-terminally fused sortase and target protein during the protein synthesis. Addition of calcium chelator BAPTA to the cell-free reaction inhibited the cleavage. In order to adapt the sortase-based purification method to the cell-free system, we next used biotin as the affinity tag. The biotinylated sortase self-cleavage purification (BISOP) method provided tag-free, highly purified proteins due to improved recovery of proteins from the resin. The N-terminal sequence analysis of the GFP produced by the BISOP method revealed that the cleavage indeed occurred at the right cleavage site. Using this method, we also successfully purified the E2 heterocomplex of USE2N and USE2v1. The c-terminal src kinase (CSK) obtained by the BISOP method showed high activity in phosphorylating the Src protein. Furthermore, we demonstrated that this method is suitable for automatically synthesizing and purifying proteins using robots.</p> <p>Conclusion</p> <p>We demonstrated that the newly developed BISOP method is very useful for obtaining high quality, tag-free recombinant proteins, produced using the cell-free system, for biochemical and structural analyses.</p

Kinetic Analysis and Prediction of Thermal Decomposition Behavior of Tertiary Pyridine Resin in the Nitrate Form

AbstractThe thermal decomposition behavior of the tertiary pyridine resin, which was used during the nuclide-separation process in the Advanced Optimization by Recycling Instructive Elements (Advanced ORIENT) cycle, was investigated in its nitrate form (TPR-NO3), in order to determine ways of preventing its runaway reaction. A thermal analysis of TPR-NO3 and an analysis of the gases produced during decomposition were employed for the purpose. In addition, the kinetics parameters were evaluated via a kinetic analysis of the empirical thermal data. Finally, the validity of the reaction model was assessed by comparing the thermal behavior predicted by the estimated reaction model with that determined by the results of a gram-scale heating test performed in our previous study. We found that, when TPR-NO3 was heated, first, nitric acid was removed. Subsequently, TPR-NO3 was oxidized by the removed nitric acid. Under the assumption that it took place an autocatalytic oxidation and nth order thermal decomposition in parallel, the thermogravimetric analysis data could be fitted very well using a nonlinear regression model. The thermal behavior of TPR-NO3 could be predicted by the reaction model determined in this study under conditions where the cooling effect owing to evaporation was ignored. In addition, the maximum temperature and time to maximum rate of a runaway reaction predicted using the determined reaction model gave the result on the side of prudence

Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate

AbstractProtein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein–protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins

Trapping-Induced Enhancement of Photocatalytic Activity on Brookite TiO<inf>2</inf> Powders: Comparison with Anatase and Rutile TiO<inf>2</inf> Powders

Brookite TiO2 is a promising material for active photocatalysts. However, the principal mechanism that determines the distinctive photocatalytic activity between anatase, rutile, and brookite TiO2 has not yet been fully elucidated. Therefore, in this work, we studied the behavior of photogenerated electrons and holes in these TiO2 powders by using femtosecond to millisecond time-resolved visible to mid-IR absorption spectroscopy. We found that most of the photogenerated electrons in brookite TiO2 are trapped at powder defects within a few ps. This electron trapping decreases the number of surviving free electrons, but it extends the lifetime of holes as well as the trapped electrons because the probability of electrons to encounter holes is decreased by this electron-trapping. As a result, the number of surviving holes increases, which is beneficial for photocatalytic oxidation. In contrast, the reactivity of electrons is decreased to some extent by trapping, but they still remain active for photocatalytic reductions. Electron trapping also takes place on anatase and rutile TiO2 powders, but the trap-depth in anatase is too shallow to extend the lifetime of holes and that of rutile is too deep than the thermal energy (kT) at room temperature for the electron-consuming reactions. As a result of the moderate depth of the electron trap in brookite, both electrons and holes are reactive for photocatalytic reductions and oxidations. These results have clearly demonstrated that the presence of an appropriate depth of the electron trap can effectively contribute to enhance the overall photocatalytic activity

Mechanical Stimulation-Induced Calcium Signaling by Piezo1 Channel Activation in Human Odontoblast Reduces Dentin Mineralization

Odontoblasts play critical roles in dentin formation and sensory transduction following stimuli on the dentin surface. Exogenous stimuli to the dentin surface elicit dentinal sensitivity through the movement of fluids in dentinal tubules, resulting in cellular deformation. Recently, Piezo1 channels have been implicated in mechanosensitive processes, as well as Ca(2+) signals in odontoblasts. However, in human odontoblasts, the cellular responses induced by mechanical stimulation, Piezo1 channel expression, and its pharmacological properties remain unclear. In the present study, we examined functional expression of the Piezo1 channel by recording direct mechanical stimulation-induced Ca(2+) signaling in dentin matrix protein 1 (DMP-1)-, nestin-, and dentin sialophosphoprotein (DSPP)-immunopositive human odontoblasts. Mechanical stimulation of human odontoblasts transiently increased intracellular free calcium concentration ([Ca(2+)](i)). Application of repeated mechanical stimulation to human odontoblasts resulted in repeated transient [Ca(2+)](i) increases, but did not show any desensitizing effects on [Ca(2+)](i) increases. We also observed a transient [Ca(2+)](i) increase in the neighboring odontoblasts to the stimulated cells during mechanical stimulation, showing a decrease in [Ca(2+)](i) with an increasing distance from the mechanically stimulated cells. Application of Yoda1 transiently increased [Ca(2+)](i). This increase was inhibited by application of Gd(3+) and Dooku1, respectively. Mechanical stimulation-induced [Ca(2+)](i) increase was also inhibited by application of Gd(3+) or Dooku1. When Piezo1 channels in human odontoblasts were knocked down by gene silencing with short hairpin RNA (shRNA), mechanical stimulation-induced [Ca(2+)](i) responses were almost completely abolished. Piezo1 channel knockdown attenuated the number of Piezo1-immunopositive cells in the immunofluorescence analysis, while no effects were observed in Piezo2-immunopositive cells. Alizarin red staining distinctly showed that pharmacological activation of Piezo1 channels by Yoda1 significantly suppressed mineralization, and shRNA-mediated knockdown of Piezo1 also significantly enhanced mineralization. These results suggest that mechanical stimulation predominantly activates intracellular Ca(2+) signaling via Piezo1 channel opening, rather than Piezo2 channels, and the Ca(2+) signal establishes intercellular odontoblast-odontoblast communication. In addition, Piezo1 channel activation participates in the reduction of dentinogenesis. Thus, the intracellular Ca(2+) signaling pathway mediated by Piezo1 channels could contribute to cellular function in human odontoblasts in two ways: (1) generating dentinal sensitivity and (2) suppressing physiological/reactional dentinogenesis, following cellular deformation induced by hydrodynamic forces inside dentinal tubules

PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation

Lentiviruses have evolved to acquire an auxiliary protein Vpx to counteract the intrinsic host restriction factor SAMHD1. Although Vpx is phosphorylated, it remains unclear whether such phosphorylation indeed regulates its activity toward SAMHD1. Here we identify the PIM family of serine/threonine protein kinases as the factors responsible for the phosphorylation of Vpx and the promotion of Vpx-mediated SAMHD1 counteraction. Integrated proteomics and subsequent functional analysis reveal that PIM family kinases, PIM1 and PIM3, phosphorylate HIV-2 Vpx at Ser13 and stabilize the interaction of Vpx with SAMHD1 thereby promoting ubiquitin-mediated proteolysis of SAMHD1. Inhibition of the PIM kinases promotes the antiviral activity of SAMHD1, ultimately reducing viral replication. Our results highlight a new mode of virus–host cell interaction in which host PIM kinases facilitate promotion of viral infectivity by counteracting the host antiviral system, and suggest a novel therapeutic strategy involving restoration of SAMHD1-mediated antiviral response

Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L

Generation of PI3P in the normally PI3P-deficient ER membrane makes the organelle a platform for autophagosome formation