Time Dependence of Tip Morphology during Cellular/Dendritic Arrayed Growth

Succinonitrile-1.9 wt pct acetone has been directionally solidified in 0.7 X 0.7-cm-square cross section pyrex ampoules in order to observe the cell/dendrite tip morphologies, not influenced by the 'wall effects', which are present during growth in the generally used thin (about 200 gm) crucibles. The tips do not maintain a steady-state shape, as is generally assumed. Instead, they fluctuate within a shape envelope. The extent of fluctuation increases with decreasing growth speed, as the micro structure changes from the dendritic to cellular. The influence of natural convection has been examined by comparing these morphologies with those grown, without convection, in the thin ampoules

Immobilization of Neocallimastix patriciarum xylanase on artificial oil bodies and statistical optimization of enzyme activity

A thermally stable and alkalophilic xylanase, XynCDBFV, from Neocallimastix patriciarum was overexpressed in Escherichia coli as a recombinant protein fused to the N-terminus of oleosin, a unique structural protein of seed oil bodies. As a result of the reconstitution of the artificial oil bodies (AOBs), the immobilization of active xylanase was accomplished. Response surface methodology (RSM) was employed for the optimization of the immobilized xylanase activity. The central composite design (CCD) and regression analysis methods were effective for determination of optimized temperature and pH conditions for the AOB-immobilized XynCDBFV. The optimal condition for the highest immobilized xylanase activity (3.93 IU/mg of total protein) was observed at 59 degrees C and pH 6.0. Further, ACB-immobilized XynCDBFV retained 50% of its maximal activity after 120 min at 60 degrees C. and it could be easily and simply recovered from the surface of the solution by brief centrifugation, and could be reused eight times while retaining more than 60% of its activity. These results proved it is a simple and effective method for direct immobilization of xylanases. (C) 2008 Elsevier Ltd. All rights reserved

Engineering lysine-rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over-expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step-wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys-rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 201

Stability of Artificial Oil Bodies constituted with Recombinant Caleosins

Caleosin is a unique calcium binding protein anchoring to the surface of seed oil bodies by its central hydrophobic domain composed of an amphiphatic alpha-helix and a proline-knot subdomain. Stable artificial oil bodies were successfully constituted with recombinant caleosin overexpressed in Escherichia coli. The stability of artificial oil bodies was slightly or severely reduced when the amphiphatic alpha-helix or proline-knot subdomain in the hydrophobic domain of caleosin was truncated. Deletion of the entire central hydrophobic domain substantially increased the solubility of the recombinant caleosin, leading to a complete loss of its capability to stabilize these oil bodies. A recombinant protein engineered with the hydrophobic domain of caleosin replaced by that of oleosin, the abundant structural protein of seed oil bodies, could stabilize the artificial oil bodies, in terms of thermo- and structural stability, as effectively as caleosin or oleosin

Simultaneous refolding, purification, and immobilization of recombinant Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase on artificial oil bodies

BACKGROUND: 1,3-1,4-beta-D-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase; EC 3.2.1.73) has been used in a range of industrial processes. As a biocatalyst, it is better to use immobilized enzymes than free enzymes, therefore, the immobilization of 1,3-1,4-beta-D-glucanase was investigated. RESULTS: A 1,3-1,4-beta-D-glucanase gene from Fibrobacter succinogenes was overexpressed in Escherichia coli as a recombinant protein fused to the N terminus of oleosin, a unique structural protein of seed oil bodies. With the reconstitution of the artificial oil bodies (AOBs), refolding, purification, and immobilization of active 1,3-1,4-beta-D-glucanase was accomplished simultaneously. Response surface modeling (RSM), with central composite design (CCD), and regression analysis were successfully applied to determine the optimal temperature and pH conditions of the AOB-immobilized 1,3-1,4-beta-D-glucanase. The optimal conditions for the highest immobilized 1,3-1,4-beta-D-glucanase activity (7.1 IU mg(-1) of total protein) were observed at 39 degrees C and pH 8.8. Furthermore, AOB-immobilized 1,3-1,4-beta-D-glucanase retained more than 70% of its initial activity after 120 min at 39 degrees C, and it was easily and simply recovered from the surface of the solution by brief centrifugation; it could be reused eight times while retaining more than 80% of its activity. CONCLUSIONS: These results indicate that the AOB-based system is a comparatively simple and effective method for simultaneous refolding, purification, and immobilization of 1,3-1,4-beta-D-glucanase. (C) 2009 Society of Chemical Industr

Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies

A gene encoding a xylanase, named xynS20, was cloned from the ruminal fungus Neocallimastix patriciarum. The DNA sequence of xynS20 revealed that the gene was 1,008 bp in size and encoded amino acid sequences with a predicted molecular weight of 36 kDa. The amino acid sequence alignment showed that the highest sequence identity (28.4%) is with insect gut xylanase XYL6805. According to the sequence-based classification, a putative conserved domain of glycosyl hydrolase family 11 was detected at the N-terminus of XynS20 and a putative conserved domain of family 1 carbohydrate-binding module (CBM) was observed at the C-terminus of XynS20. An Asn-rich linker sequence was found between the N-terminal catalytic domain and the C-terminal CBM of XynS20. To examine the activity of the gene product, xynS20 gene was cloned as an oleosin-fused protein, expressed in Escherichia coli, affinity-purified by formation of artificial oil bodies, released from oleosin by intein-mediated peptide cleavage, and finally harvested by concentration of the supernatant. The specific activity of purified XynS20 toward oat spelt xylan was 1,982.8 U mg(-1). The recombinant XynS20 was stable in the mild acid pH range from 5.0 to 6.0, and the optimum pH was 6.0. The optimal reaction temperature of XynS20 was 45 degrees C; at temperatures below 30 and above 55 degrees C, enzyme activity was less than 50% of that at the optimal temperature

Cloning and expression of a seed-specific metallothionein-like protein from sesame

A cDNA clone, SiMT encoding an Ec type of metallothionein (MT)-like protein, was isolated from maturing seeds of sesame (Sesamum indicum L.), and its deduced protein sequence shared 47-65% similarity to other known Ec type of MT-like proteins with three highly conserved cysteine-rich segments. The transcript of SiMT was exclusively accumulated in maturing seeds from two weeks after flowering to the end of seed maturation. The results of a southern blot analysis suggested that one SiMT and one SiMT-like gene were present in the sesame genome. Recombinant SiMT fused with glutathione-S-transferase (GST) was over-expressed in Escherichia coli, and purified to homogeneity by affinity chromatography. Recombinant SiMT released from GST was harvested after factor Xa cleavage. Migration of the recombinant SiMT during SDS-PAGE was accelerated when its binding metal ions were depleted by EDTA. The metal-binding capability of recombinant SiMT was measured by inductively-coupled plasma atomic emission spectrometry. Our results show that the recombinant SiMT could trap zinc or copper ions, but not manganese ions, with a stoichiometric ratio (metal ion/SiMT) of approximately 2

Ribosome inactivating protein B-chain induces osteoclast differentiation from monocyte/macrophage lineage precursor cells

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between lectins and their receptors. A type-2 ribosome inactivating protein consists of an A chain and a B chain. The glycosylated B chain binds specifically to galactose moieties of sugar molecules. In this study we showed that the recombinant ribosome inactivating protein B-chain (rRBC) could induce osteoclast formation from human monocytes and murine RAW264.7 macrophages. Tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays demonstrated that differentiation of osteoclast-like cells was induced in the presence of rRBC in a dose-dependent manner. The rRBC-induced osteoclast differentiation was independent of caspase activation and apoptosis induction activity; however, rRBC-induced osteoclastogenesis was dependent on activation of NF-kappa B, ERK1/2, and p38 MAP kinase. Thus, our data demonstrated that rRBC induced osteoclast differentiation through a non-apoptotic signaling pathway. In addition to triggering apoptosis, the rRBC also induced osteoclast differentiation. According to this study, a novel role is proposed for rRBC in regulating osteoclast differentiation and in osteoimmunology. (C) 2011 Elsevier Inc. All rights reserved