Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.     

Construction of crystal structures of metal(II)-benzoates (M = Mn, Ni, Co, Cu, Zn, and Cd) and 1,2-bis(4-pyridyl)ethane: Effects of metal coordination modes and their catalytic activities

Five polymeric metal(II)-benzoate complexes of formula [Mn(O₂CPh)₂(CH₃OH)₂(bpa)]_n (1-Mn), [Co(O₂CPh)₂(H₂O)(bpa)_1.5]_n (2-Co), [Ni(O₂CPh)₂(H₂O)(bpa)_1.5]_n (3-Ni), [Cu(O₂CPh)₂(CH₃OH)₂(bpa)]_n (4-Cu), and [Cd(O₂CPh)₂(bpa)_1.5]_n (6-Cd) have been synthesized and characterized (bpa = 1,2-bis(4-pyridyl)ethane). They showed two kinds of structures: parallelogram-like two-dimensional sheets for Co, Ni, and Cd, and one-dimensional chains for Mn, Cu, and Zn. Since similar structures provide similar coordination geometries, the structures depend on the coordination geometries of metal ions. The compounds 1-Mn, 2-Co, 4-Cu, 5-Zn, and 6-Cd have catalyzed efficiently the transesterification of a variety of esters, while 3-Ni has displayed a very slow conversion. The reactivity of catalyst 6-Cd containing Cd ion, well known as an inert metal ion for the ligand substitution, was found to be comparable to that of 5-Zn. The reactivities of the compounds used in this study are in the order of 5-Zn > 6-Cd > 1-Mn > 4-Cu > 2-Co ? 3-Ni, indicating that the non-redox metal-containing compounds (5-Zn and 6-Cd) show better activity than the redox-active metal-containing compounds (1-Mn, 4-Cu, 2-Co, and 3-Ni).     

Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway

Background  

MPG is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. In this work, we examined the efficacy of MPG as an N-terminal tag in a fusion protein to deliver a protein cargo and its mechanism of transduction.     

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.     

Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine.

One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxidase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis induced by activated phenoloxidase. In this study, to isolate and characterize proteins involved in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution), specifically showing phenoloxidase activity in the presence of Ca2+ and beta-1, 3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 column. When G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca2+ and beta-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, phenoloxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160-kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase induced melanin synthesis. Furthermore, when the purified 160-kDa melanization-engaging protein was added to a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conclusion that the 160-kDa vitellogenin-like protein is involved in arthropod melanin synthesis.     

Centriolin,a centriole-appendage protein,regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.