Searching sequence space for high-affinity binding peptides using ribosome display

We present the construction of a synthetic library based on the scaffold of bovine heart fatty acid-binding protein (FABP) with 1.1x10(14) independent members. Ribosome display was applied to select streptavidin-binding peptides in vitro from 2x10(13) molecules of the library each encoding FABP with 15 contiguous random amino acid residues at its N terminus. The selection yielded several different binding peptides. The best binder possessed a dissociation constant as low as 4nM and, in contrast to the previously isolated peptides, contained no HPQ motif. A substitution analysis enabled shortening of the 15-mer peptide and revealed a 9-mer variant with a dissociation constant of 17nM, which is a 1000-fold increase of affinity compared to the already known peptides of this size. This high-affinity binding peptide in combination with the whole set of streptavidin conjugates should be an extremely useful tool for the detection and purification of recombinant proteins.     

Series of vectors to evaluate the position and the order of two different affinity tags for purification of protein complexes

A series of protein expression vectors with dual-affinity tags has been developed. With these constructed vectors, FLAG and hexahistidine tags were fused to a given protein at either the N- or the C-terminal ends or both, for a total of six combinations. Three auxotrophy markers were introduced into each construct, thus yielding 18 different vectors. These vectors allow evaluation of different positions and orders of two different tags. To confirm the efficacy of these vectors, we purified a histone acetyltransferase (Esa1p)-containing complex. First, an appropriate position of the tags was selected through small-scale purification. Next, large-scale purification was done for the selected construct, yielding an Esa1p-containing complex that was comparable to an Esa1p-containing complex (NuA4) obtained by a conventional activity-based purification. These vectors provide a convenient way to select the best position of tags for efficient purification of protein complexes also applicable in proteomics studies.     

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.     

Blocking the dengue virus 2 infections on BHK-21 cells with purified recombinant dengue virus 2 E protein expressed in Escherichia coli

Dengue viruses (DVs) are mosquito-borne infectious pathogens. They have become an expanding public health problem in the tropics and subtropics. The dengue envelope (E) protein is one of the viral structure proteins responsible mainly for the virus attachment and entry onto host cells. It is also the major immunogen for virus neutralization. In this study, we have constructed a recombinant plasmid expressing a truncated E protein of DV-2 virus PL046 strain. The C-terminal hydrophobic domain of the E protein was removed and replaced with the sequence of S peptide to facilitate expression and purification. When expressed in Escherichia coli, the recombinant E proteins were found to be in the form of aggregated state. Through denaturation and dialysis processes, the receptor-interacting function of the purified recombinant E proteins was maintained, which was demonstrated by its ability to inhibit the DV-2 plaque-forming efficiency on mammalian BHK-21 host cells.     

Use of epitope tags for routine analysis of transgene expression

Peptide and RNA epitope tags as tools for routine analysis of transgene expression and protein accumulation in transformed plant cell cultures was evaluated using three genes that encode very structurally and functionally different proteins. A T7 peptide was introduced at the amino- and carboxyl-termini of phosphinothricin-N-acetyl transferase and avidin and at the carboxyl-terminus of galactose oxidase. An RNA sequence that forms a higher order structure that is recognized by antibodies raised against the FLAG peptide was separately introduced into the 3 nontranslated region of these genes. Constructs were introduced into maize cell cultures using particle bombardment and transgene expression, protein accumulation, protein function and presence of the tags in RNA and/or protein as appropriate were evaluated in up to approximately 25 culture lines per construct. Results indicate that, while there will likely always be a need for some empirical evaluation of any tag-protein combination, introduction of the peptide tag at the amino-terminus was generally more successful than was incorporation at the carboxyl-terminus. RNA tags show promise for this purpose, but routine application will require development of a very sensitive immunoassay.Both of these authors contributed equally to this work and should be recognized as first authors.     

Unravelling cell wall formation in the woody dicot stem

Populus is presented as a model system for the study of wood formation (xylogenesis). The formation of wood (secondary xylem) is an ordered developmental process involving cell division, cell expansion, secondary wall deposition, lignification and programmed cell death. Because wood is formed in a variable environment and subject to developmental control, xylem cells are produced that differ in size, shape, cell wall structure, texture and composition. Hormones mediate some of the variability observed and control the process of xylogenesis. High-resolution analysis of auxin distribution across cambial region tissues, combined with the analysis of transgenic plants with modified auxin distribution, suggests that auxin provides positional information for the exit of cells from the meristem and probably also for the duration of cell expansion. Poplar sequencing projects have provided access to genes involved in cell wall formation. Genes involved in the biosynthesis of the carbohydrate skeleton of the cell wall are briefly reviewed. Most progress has been made in characterizing pectin methyl esterases that modify pectins in the cambial region. Specific expression patterns have also been found for expansins, xyloglucan endotransglycosylases and cellulose synthases, pointing to their role in wood cell wall formation and modification. Finally, by studying transgenic plants modified in various steps of the monolignol biosynthetic pathway and by localizing the expression of various enzymes, new insight into the lignin biosynthesis in planta has been gained.     

Chromosomal assignments for porcine genes encoding enzymes in hepatic metabolic pathways

Increasing the number of mapped genes will facilitate (1) the identification of potential candidate genes for a trait of interest within quantitative trait loci regions and (2) comparative mapping. The metabolic activities of the liver are essential for providing fuel to peripheral organs, for regulation of amino acid, carbohydrate and lipid metabolism and for homoeostasis of vitamins, minerals and electrolytes. We aimed to identify and map genes coding for enzymes active in the liver by somatic cell genetics in order to contribute to the improvement of the porcine gene map. We mapped 28 genes of hepatic metabolic pathways including six genes whose locations could be confirmed and 22 new assignments. Localization information in human was available for all but one gene. In total 24 genes were assigned to in the expected chromosomal regions on the basis of the currently available information on the comparative human and pig map while for four genes our results suggest a new correspondence or extended regions of conservation between porcine and human chromosomes.     

The P1' specificity of tobacco etch virus protease

Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the tobacco etch virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.     

Automated whole mount localisation techniques for plant seedlings

Plant biology is currently experiencing a growing demand for easy and reliable mRNA and protein localisation techniques. Here, we present novel whole mount in situ hybridisation and immunolocalisation protocols, suitable to localise mRNAs and proteins in Arabidopsis seedlings. We demonstrate that these methods can be used in different organs of Arabidopsis seedlings as well as in other plant species. In order to achieve better reproducibility and higher throughput, we modified these protocols for automation to be performed by a liquid handling robot. In addition, we show that other procedures such as reporter enzyme assays and tissue clearing can be similarly automated. We present examples of application of our protocols including mRNA localisation and proteins and epitope tag (co)localisations which demonstrate that these methods provide reliable and versatile tools for expression, localisation and anatomical studies in plants.