Structure and dimerization of translation initiation factor aIF5B in solution.

Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.

Structural transitions of translation initiation factor IF2 upon GDPNP and GDP binding in solution.

Three protein factors ensure rapid and accurate initiation of translation in bacteria. Translation initiation factor IF2 is a ribosome-dependent GTPase, which is important for correct positioning of initiator tRNA on the 30S subunit as well as ribosomal subunit joining. The solution structure of the free C-terminal part of IF2 (IF2C, comprising domains IV to VI-2) was previously determined by small-angle X-ray scattering (SAXS) [Rasmussen, L.

Expressivity tag: a novel tool for increased expression in Escherichia coli.

Protein expression in Escherichia coli is rarely trivial as low expression and insolubility are common problems. In this work we define a fusion partner, which increases expression levels similarly to the distinct function of solubility and affinity tags. This type of fusion tag we term an expressivity tag.

Solution structure of C-terminal Escherichia coli translation initiation factor IF2 by small-angle X-ray scattering.

Initiation of protein synthesis in bacteria involves the combined action of three translation initiation factors, including translation initiation factor IF2. Structural knowledge of this bacterial protein is scarce. A fragment consisting of the four C-terminal domains of IF2 from Escherichia coli was expressed, purified, and characterized by small-angle X-ray scattering (SAXS), and from the SAXS data, a radius of gyration of 43 +/- 1 A and a maximum dimension of approximately 145 A were obtained for the molecule.

Production and epitope characterization of mAbs specific for translation factor IF1.

Initiation of protein synthesis in bacteria relies on the presence of three translation initiation factors, of which translation initiation factor IF1 is the smallest having a molecular weight of only 8.2kDa. In addition to its function in this highly dynamic process, the essential IF1 protein also functions as an RNA chaperone.

Enantioselective proteins: selection, binding studies and molecular modeling of antibodies with affinity towards hydrophobic BINOL derivatives.

In this paper, the initial steps towards the design of novel artificial metalloenzymes that exploit proteins as a second coordination sphere for traditional metal-ligand catalysis are described. Phage display was employed to select and study antibody fragments capable of recognizing hydrophobic BINOL derivatives designed to mimic BINAP, a widely used ligand in asymmetric metal-catalyzed reactions. The binding affinities of the selected antibodies towards a series of haptens were evaluated by using ELISA assays.

Hitting bacteria at the heart of the central dogma: sequence-specific inhibition.

An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long, regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results.

Salinity and temperature effects on accessibility of soluble and cross-linked insoluble xylans to endo-xylanases.

Different responses to salinity were observed for an extremely halotolerant endo-xylanase when assayed with soluble birchwood glucoronoxylan and cross-linked dyed insoluble birchwood glucoronoxylan. Shrinking of insoluble xylan particles due to increased ionic strength is proposed as the explanation. Temperature affected the xylanase activity measurement on the insoluble xylan greatly, likely due to increased enzyme accessible surface of the substrate at high temperatures.

Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling.

We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23) followed by expression in an rpL23 deficient strain of E.

Initiation of protein synthesis in bacteria.

Valuable information on translation initiation is available from biochemical data and recently solved structures. We present a detailed description of current knowledge about the structure, function, and interactions of the individual components involved in bacterial translation initiation. The first section describes the ribosomal features relevant to the initiation process.

Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli.

Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing.

Advanced genetic strategies for recombinant protein expression in Escherichia coli.

Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis.

Soluble expression of aggregating proteins by covalent coupling to the ribosome.

Ribosomes are extremely soluble ribonucleoprotein complexes. Heterologous target proteins were fused to ribosomal protein L23 (rpL23) and expressed in an rpL23 deficient Escherichia coli strain. This enabled the isolation of 70S ribosomes with covalently bound target protein.

A favorable solubility partner for the recombinant expression of streptavidin.

Recombinant streptavidin is extremely difficult to express at high levels in the cytoplasm of Escherichia coli without the formation of inclusion bodies. Fusing a solubility enhancing partner to an aggregation prone protein is a widely used tool to circumvent inclusion body formation. Here, we use streptavidin as a target protein to test the properties of N-terminal fragments of translation initiation factor IF2 from E.

The N-terminal domain (IF2N) of bacterial translation initiation factor IF2 is connected to the conserved C-terminal domains by a flexible linker.

Bacterial translation initiation factor IF2 is a multidomain protein that is an essential component of a system for ensuring that protein synthesis begins at the correct codon within a messenger RNA. Full-length IF2 from Escherichia coli and seven fragments of the protein were expressed, purified, and characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) methods. Interestingly, resonances of the 6 kD IF2N domain located at the extreme N terminus of IF2 can be clearly identified within the NMR spectra of the full-length 97-kD protein.

Dialysis strategies for protein refolding: preparative streptavidin production.

We have investigated different dialysis strategies for the refolding of recombinant streptavidin, and present a novel dialysis setup featuring gradual dilution dialysis and continuous protein feeding into a dialysis sack. A denaturing dialysis buffer is exchanged gradually by dilution with refolding buffer and it is demonstrated that the refolding yield can be increased from 45 to 75% by lowering the dilution rate. In addition, continuous feeding of protein to the dialysis sack increases the yield by 5 to 10%.

Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium.

The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.

Production of recombinant thermostable proteins expressed in Escherichia coli: completion of protein synthesis is the bottleneck.

Heterologous expression and high yield purification of proteins are frequently required for structural and functional investigations. Purification of recombinant thermostable proteins is essentially trivial since unwanted mesophilic host protein can efficiently be removed by heat denaturation. However, heterologous expression in E.

A conserved structural motif at the N terminus of bacterial translation initiation factor IF2.

The 18-kDa Domain I from the N-terminal region of translation initiation factor IF2 from Escherichia coli was expressed, purified, and structurally characterized using multidimensional NMR methods. Residues 2-50 were found to form a compact subdomain containing three short beta-strands and three alpha-helices, folded to form a betaalphaalphabetabetaalpha motif with the three helices packed on the same side of a small twisted beta-sheet. The hydrophobic amino acids in the core of the subdomain are conserved in a wide range of species, indicating that a similarly structured motif is present at the N terminus of IF2 in many of the bacteria.

Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-sensitive growth of Escherichia coli.

The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24.

Structural requirements of the mRNA for intracistronic translation initiation of the enterobacterial infB gene.

Background: The gene infB encodes the prokaryotic translation initiation factor IF2, a central macromolecular component in the formation of the ribosomal 70S initiation complex. In Escherichia coli, infB encodes three forms of IF2: IF2alpha, IF2beta and IF2gamma. The expression of IF2beta and IF2gamma is a tandem translation from intact infB mRNA and not merely a translation of post-transcriptionally truncated mRNA.

Investigation of the translation-initiation factor IF2 gene, infB, as a tool to study the population structure of Streptococcus agalactiae.

The sequence of infB, encoding the prokaryotic translation-initiation factor 2 (IF2), was determined in eight strains of Streptococcus agalactiae (group B streptococcus) and an alignment revealed limited intraspecies diversity within S. agalactiae. The amino acid sequence of IF2 from S.