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Recombinant techniques for target protein production have been rapidly established and widely utilised in today's biological research. Nevertheless, methods for membrane protein production have yet to be developed, since membrane proteins generally tend to be expressed at low levels, easily aggregated, and/or even toxic to their host cells. Here we report that a GFP-tagging technique can be applied for the stable production of membrane proteins that are toxic to their host cells when overexpressed, paving the way for future advances in membrane protein biochemistry and drug development. [ABSTRACT FROM AUTHOR]

MALDI−TOF MS has developed rapidly into an essential analytical tool for the life sciences. Cinnamic acid derivatives are generally employed in routine molecular weight determinations of intact proteins using MALDI−TOF MS. However, a protein of interest may precipitate when mixed with matrix solution, perhaps preventing MS detection. We herein provide a simple approach to enable the MS detection of such precipitated protein species by means of a “direct deposition method” -- loading the precipitant directly onto the sample plate. It is thus expected to improve routine MS analysis of intact proteins. [ABSTRACT FROM AUTHOR]

Glutaminyl-transfer RNA (Gln-tRNAGln) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNAGln by the heterodimeric Glu-tRNAGln amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNAGln at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNAGln mutants characterized the catalytic centers for the enzyme's three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom-long channel for ammonia transport connects the active sites in GatD and GatE. tRNAGln recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code. [ABSTRACT FROM AUTHOR]

Gelatin, sourced from collagen, is an acid-, alkali- or enzymatically hydrolysed product obtained from animal skins and bones. Gelatin has been widely used for the manufacture of various cultural objects, e.g. as a water-soluble binder for dissolving pigments, and as a glue for musical instruments and traditional crafts along with human history. The identification of animal species in gelatin, hence, could provide a critical clue for understanding human history including lifestyles, the culture and the technologies. However, there has been no valid method established to date for identifying the animal species from traditional gelatins. We herein report that the nucleic acids contents (dsDNA, ssDNA and miRNA) from commercially-available gelatins manufactured according to classical procedures (wanikawa) exhibited much higher (about 10 times) than those from modern gelatins made through an industrialised process (yonikawa), suggesting that DNA analysis using the gelatins from cultural assets could be substantially feasible. Moreover, targeting not only commercially available niwaka but also Ukiyo-e, Japanese classical art manufactured through woodblock printings, we here illustrate partial successes in the animal species identification coupled with DNA barcoding technique, hopefully paving the way for scientifically more reliable animal species identifications of archaeological specimens made with a gelatin component. [ABSTRACT FROM AUTHOR]

Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli . A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1 M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5 M l -arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l -arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method. [ABSTRACT FROM AUTHOR]

Summary: The U5 small nuclear ribonucleoprotein particle (snRNP) helicase Brr2 disrupts the U4/U6 small nuclear RNA (snRNA) duplex and allows U6 snRNA to engage in an intricate RNA network at the active center of the spliceosome. Here, we present the structure of yeast Brr2 in complex with the Jab1/MPN domain of Prp8, which stimulates Brr2 activity. Contrary to previous reports, our crystal structure and mutagenesis data show that the Jab1/MPN domain binds exclusively to the N-terminal helicase cassette. The residues in the Jab1/MPN domain, whose mutations in human Prp8 cause the degenerative eye disease retinitis pigmentosa, are found at or near the interface with Brr2, clarifying its molecular pathology. In the cytoplasm, Prp8 forms a precursor complex with U5 snRNA, seven Sm proteins, Snu114, and Aar2, but after nuclear import, Brr2 replaces Aar2 to form mature U5 snRNP. Our structure explains why Aar2 and Brr2 are mutually exclusive and provides important insights into the assembly of U5 snRNP. [Copyright &y& Elsevier]

Abstract: Two kinds of layer silicate powder, Micromica and chlorite, were used to aid protein crystallization by the addition to hanging drops. Using appropriate crystallization buffers, Micromica powder facilitated crystal growth speed for most proteins tested in this study. Furthermore, the addition of Micromica powder to hanging drops allowed the successful crystallization of lysozyme, catalase, concanavalin A, and trypsin even at low protein concentrations and under buffer conditions that otherwise would not generate protein crystals. Except for threonine synthase and apoferritin, the presence of chlorite delayed crystallization but induced the formation of large crystals. X-ray analysis of thaumatin crystals generated by our novel procedure gave better quality data than did that of crystals obtained by a conventional hanging drop method. Our results suggest that the speed of crystal growth and the quality of the corresponding X-ray data may be inversely related, at least for the formation of thaumatin crystals. The effect of Micromica and chlorite powders and the application of layer silicate powder for protein crystallization are discussed. [Copyright &y& Elsevier]