RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20–2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5′-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

hGM-CSF (human granulocytemacrophage colony stimulating factor) is a cytokine secreted by many cell types. Its characters are suitable for vaccine adjuvant such as ability to stimulate survival, differentiation and enhancement the functions of antigen-presenting cells. This cytokine is also a chemoattractant for monocytes and neutrophils to the infected sites, stimulates the expression of several cytokines like IL-1, IL-6, TNF, which are essential for B and T lymphocyte differentiation. However, hGM-CSF has some drawbacks for being an adjuvant candidate due to its easy degradation, toxicity at high concentration and low-dose requirement for therapeutic effect. Drugs delivery system using chitosan can overcome these disadvantages of hGM-CSF. In this present study, chitosan particles were prepared and evaluated the absorption and release of human hGM-CSF. Firstly, the activity of hGM CSF was evaluated by proliferation bioassay using TF-1 cell line. Afterward, chitosan particles were prepared by ionic gelation method and were examined for its toxicity on TF‑1 cell line. After protein absorbance onto chitosan particles, the release capacity and in vitro protection of chitosan for hGM-CSF were assessed. The result showed that hGM-CSF had an ED50 value of 106 pg/mL. The synthesized chitosan particles had an average diameter of 24.5 nm and were nontoxic. Based on the results of SDS-PAGE and Bradford, the adsorption efficiency of hGM‑CSF onto chitosan particles reached 99 % and chitosan has the ability to release hGM-CSF and protects it from hydrolysis of trypsin. In conclusion, the synthesized chitosan beads absorbed and released hGMCSF with its activity remained. This result provides the evidence for further in vivo researches.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein, belongs to the family of colony stimulating factors (CSFs) that regulate proliferation and differentiation of hematopoietic cells. Recombinant hGM-CSF (rhGM-CSF) is one of FDA-appoved, therapeutic recombinant proteins that have been successfully used to treat many diseases like neutropenia, leukemia, or in combination with chemical therapy, etc. In this study, we reported the production of rhGM-CSF in methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. The gene hgm-csf encoding for hGM-CSF comprising of 415 bps was isolated using PCR reaction with two primers hGM-F and hGM-R bearing XhoI and NotI restriction sites, respectively. After double digesting with these enzymes, the hgm-csf fragment was cloned into pPICZαA shuttle vector, between the XhoI and NotI sites. Recombinant vector containing the gene hgm-csf, termed pPICZαA/hGMCSF, under the control of promoter AOX has the ability to express hGM-CSF in P. pastoris. The accuracy of cloning strategy was confirmed by digestion with REs, PCR and sequencing. Sequencing alignment showed that the cloned gene completely homologous to those published on Genebank. SacI linearized pPICZαA/hGM-CSF was transformed into P. pastoris X33 strain to establish the recombinant P. pastoris X33::hgm-csf. In methanol-contained, inducing BMMY medium, the recombinant X33 cells expressed and secreted hGM-CSF into the supernatant. The secreted hGM-CSF migrated as a band at about 20 kDa, a diffuse band at the range of 29 to 35 kDa, indicating differentially glycosylated forms, and a band at 14,7kDa which is a nonglycosylated form on SDS-PAGE gel. This result was confirmed by Western blot with specific antibodies.