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. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Historical profiling of maize duplicate genes sheds light on the evolution of C4 photosynthesis in grasses

C4 plants evolved from C3 plants through a series of complex evolutionary steps. On the basis of the evolution of key C4 enzyme genes, the evolution of C4 photosynthesis has been considered a story of gene/genome duplications and subsequent modifications of gene function. If whole-genome duplication has contributed to the evolution of C4 photosynthesis, other genes should have been duplicated together with these C4 genes. However, which genes were co-duplicated with C4 genes and whether they have also played a role in C4 evolution are largely unknown. In this study, we developed a simple method to characterize the historical profile of the paralogs of a gene by tracing back to the most recent common ancestor (MRCA) of the gene and its paralog(s) and then counting the number of paralogs at each MRCA. We clustered the genes into clusters with similar duplication profiles and inferred their functional enrichments. Applying our method to maize, a familiar C4 plant, we identified many genes that show similar duplication profiles with those of the key C4 enzyme genes and found that the functional preferences of the C4 gene clusters are not only similar to those identified by an experimental approach in a recent study but also highly consistent with the functions required for the C4 photosynthesis evolutionary model proposed by S.F. Sage. Some of these genes might have co-evolved with the key C4 enzyme genes to increase the strength of C4 photosynthesis. Moreover, our results suggested that most key C4 enzyme genes had different origins and have undergone a long evolutionary process before the emergence of C4 grasses (Andropogoneae), consistent with the conclusion proposed by previous authors.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Lipid Droplet-Binding Protein TIP47 Regulates Hepatitis C Virus RNA Replication through Interaction with the Viral NS5A Protein

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.     

Ser/Thr Kinase-Like Protein of Nicotiana benthamiana Is Involved in the Cell-to-Cell Movement of Bamboo mosaic virus

To investigate the plant genes affected by Bamboo mosaic virus (BaMV) infection, we applied a cDNA-amplified fragment length polymorphism technique to screen genes with differential expression. A serine/threonine kinase-like (NbSTKL) gene of Nicotiana benthamiana is upregulated after BaMV infection. NbSTKL contains the homologous domain of Ser/Thr kinase. Knocking down the expression of NbSTKL by virus-induced gene silencing reduced the accumulation of BaMV in the inoculated leaves but not in the protoplasts. The spread of GFP-expressing BaMV in the inoculated leaves is also impeded by a reduced expression of NbSTKL. These data imply that NbSTKL facilitates the cell-to-cell movement of BaMV. The subcellular localization of NbSTKL is mainly on the cell membrane, which has been confirmed by mutagenesis and fractionation experiments. Combined with the results showing that active site mutation of NbSTKL does not change its subcellular localization but significantly affects BaMV accumulation, we conclude that NbSTKL may regulate BaMV movement on the cell membrane by its kinase-like activity. Moreover, the transient expression of NbSTKL does not significantly affect the accumulation of Cucumber mosaic virus (CMV) and Potato virus X (PVX); thus, NbSTKL might be a specific protein facilitating BaMV movement.     

Does Temporal Integration Occur for Unrecognizable Words in Visual Crowding?

Visual crowding—the inability to see an object when it is surrounded by flankers in the periphery—does not block semantic activation: unrecognizable words due to visual crowding still generated robust semantic priming in subsequent lexical decision tasks. Based on the previous finding, the current study further explored whether unrecognizable crowded words can be temporally integrated into a phrase. By showing one word at a time, we presented Chinese four-word idioms with either a congruent or incongruent ending word in order to examine whether the three preceding crowded words can be temporally integrated to form a semantic context so as to affect the processing of the ending word. Results from both behavioral (Experiment 1) and Event-Related Potential (Experiment 2 and 3) measures showed congruency effect in only the non-crowded condition, which does not support the existence of unconscious multi-word integration. Aside from four-word idioms, we also found that two-word (modifier + adjective combination) integration—the simplest kind of temporal semantic integration—did not occur in visual crowding (Experiment 4). Our findings suggest that integration of temporally separated words might require conscious awareness, at least under the timing conditions tested in the current study.     

Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem

A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K(i) values of 1.18 x 10(-10) M and 9.53 x 10(-11) M, respectively. The recombinant cystatins were found to be thermally stable up to 60 degrees C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.