Expression of Recombinant Human Octamer-Binding Transcription Factor 4 in Rice Suspension Cells.

The rice cell suspension culture system is a good way to produce recombinant human proteins, owing to its high biosafety and low production cost. Human Octamer-binding Transcription Factor 4 (Oct4) is a fundamental transcription factor responsible for maintaining human pluripotent embryonic stem cells. Recombinant Oct4 protein has been used to induce pluripotent stem cells. In this study, recombinant Oct4 proteins are produced via a sugar starvation-inducible αAmy3/RAmy3D promoter-signal peptide-based rice recombinant protein expression system. Oct4 mRNAs accumulate in the transgenic rice suspension cells under sugar starvation. The Oct4 recombinant protein is detected in the transgenic rice suspension cells, and its highest yield is approximately 0.41% of total cellular soluble proteins after one day of sugar starvation. The rice cell-synthesized recombinant human Oct4 protein show DNA-binding activity in vitro, which implies that the protein structure is correct for enabling specific binding to the target DNA motif.

Random-Coil Behavior of Chemically Denatured Topologically Knotted Proteins Revealed by Small-Angle X-ray Scattering.

Recent studies on the mechanisms by which topologically knotted proteins attain their natively knotted structures have intrigued theoretical and experimental biophysicists. Of particular interest is the finding that YibK and YbeA, two small trefoil knotted proteins, remain topologically knotted in their chemically denatured states. Using small-angle X-ray scattering (SAXS), we examine whether these chemically denatured knotted proteins are different from typical random coils. By revisiting the scaling law of radius of gyration (Rg) as a function of polypeptide chain length for chemically denatured proteins and natively folded proteins, we find that the chemically denatured knotted proteins in fact follow the same random coil-like behavior, suggesting that the formation of topological protein knots do not necessarily require global compaction while the loosely knotted polypeptide chains are capable of maintaining the correct chirality without defined secondary or tertiary structures.

Backbone NMR assignments of a topologically knotted protein in urea-denatured state.

YbeA is a 3-methylpseudoridine methyltransferase from Escherichia coli that forms a stable homodimer in solution. It is one of the deeply trefoil 31 knotted proteins, of which the knot encompasses the C-terminal helix that threads through a long loop. Recent studies on the knotted protein folding pathways using YbeA have suggested that the protein knot remains present under chemically denaturing conditions. Here, we report (1)H, (13)C and (15)N chemical shift assignments for urea-denatured YbeA, which will serve as the basis for further structural characterisations using solution state NMR spectroscopy with paramagnetic spin labeled and partial alignment media.

Backbone 1H, 13C and 15N assignments of YibK and avariant containing a unique cysteine residue at C-terminus in 8 M urea-denatured states [corrected].

YibK is a tRNA methyltransferase from Haemophilus influenzae, which forms a stable homodimer in solution and contains a deep trefoil 31 knot encompassing the C-terminal helix that threads through a long loop. It has been a model system for investigating knotted protein folding pathways. Recent data have shown that the polypeptide chain of YibK remains loosely knotted under highly denaturing conditions. Here, we report (1)H, (13)C and (15)N chemical shift assignments for YibK and its variant in the presence of 8 M urea. This work forms the basis for further analysis using NMR techniques such as paramagnetic relaxation enhancement, residual dipolar couplings and spin-relaxation dynamics analysis.

NMR relaxation and structural elucidation of peptides in the presence and absence of trifluoroethanol illuminates the critical molecular nature of integrin αvß6 ligand specificity.

Integrin αvß6 is an important emerging target for both imaging and therapy of cancer that requires specific ligands based on Arg-Gly-Asp (RGD) peptides. There remains little correlation between integrin-RGD ligand specificity despite studies suggesting an RGD-turn-helix ligand motif is required. Here, we describe the application of 15N NMR relaxation analyses and structure determination of αvß6 peptide ligands in the presence and absence of trifluoroethanol (TFE) to identify their critical molecular nature that influences specificity, interaction and function. Two linear peptides; one known to demonstrate αvß6 specificity (FMDV2) and the other based on a natural RGD ligand (LAP2), were compared to two additional peptides based on FMDV2 but cyclised in different positions using a disulphide bond (DBD1 and DBD2). The cyclic adaptation in DBD1 produces a significant alteration in backbone dynamic properties when compared to FMDV2; a potential driver for the loss in αvß6 specificity by DBD1. The importance of ligand dynamics are highlighted through a comprehensive reduced spectral density and ModelFree analysis of peptide 15N NMR relaxation data and suggest αvß6 specificity requires the formation of a structurally rigid helix preceded by a RGD motif exhibiting slow internal motion. Additional observations include the effect of TFE/water viscosity on global NMR dynamics and the advantages of using spectral density NMR relaxation data to estimate correlation times and motional time regimes for peptides in solution.

Multiple genome sequences alignment algorithm based on coding regions.

Multiple Sequence Alignment (MSA) is the computational biology tool for facilitating the study of DNA homology, phylogeny determinations and conserved motifs. Many MSA methods have been presented to align protein, DNA, and RNA sequences successfully but not for coding region sequences. Therefore, we propose a heuristic alignment method, CORAL-M, for multiple genome sequences, especially for coding regions. CORAL-M adopts a codon-based probabilistic filtration model and the local optimal alignment solution to align multiple genome sequences in linear time. The experimental results presents that CORAL-M can find more potential function sites than that of other commonly used tools by aligning Enterovirus strains.

GeneAlign: a coding exon prediction tool based on phylogenetical comparisons.

GeneAlign is a coding exon prediction tool for predicting protein coding genes by measuring the homologies between a sequence of a genome and related sequences, which have been annotated, of other genomes. Identifying protein coding genes is one of most important tasks in newly sequenced genomes. With increasing numbers of gene annotations verified by experiments, it is feasible to identify genes in the newly sequenced genomes by comparing to annotated genes of phylogenetically close organisms. GeneAlign applies CORAL, a heuristic linear time alignment tool, to determine if regions flanked by the candidate signals (initiation codon-GT, AG-GT and AG-STOP codon) are similar to annotated coding exons. Employing the conservation of gene structures and sequence homologies between protein coding regions increases the prediction accuracy. GeneAlign was tested on Projector dataset of 491 human-mouse homologous sequence pairs. At the gene level, both the average sensitivity and the average specificity of GeneAlign are 81%, and they are larger than 96% at the exon level. The rates of missing exons and wrong exons are smaller than 1%. GeneAlign is a free tool available at http://genealign.hccvs.hc.edu.tw.