A method for measuring binding constants using unpurified in vivo biotinylated ligands

Obtaining accurate kinetics and steady-state binding constants for biomolecular interactions normally requires pure and homogeneous protein preparations. Furthermore, in many cases, one of the ligands must be labeled. Over the past decade, several technologies have been introduced that allow for the measurement of kinetics constants for multiple different interactions in parallel. One such technology is bio-layer interferometry (BLI), which has been used to develop systems that can measure up to 96 biomolecular interactions simultaneously. However, despite the ever-increasing throughput of the tools available for measuring protein–protein interactions, the preparation of pure protein still remains a bottleneck in the process of producing high-quality kinetics data. Here, we show that high-quality binding data can be obtained using soluble lysate fractions containing protein that has been biotinylated in vivo using BirA and then applied to BLI sensors without further purification. Furthermore, we show that BirA ligase does not necessarily need to be co-overexpressed with the protein of interest for biotinylation of the biotin acceptor peptide to occur, suggesting that the activity of endogenous BirA in Escherichia coli is sufficient for producing enough biotinylated protein for a binding experiment.     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

Improvement in the bleaching of kraft pulp with xylanase from Penicillium crustosum FP 11 isolated from the Atlantic forest

Xylanase produced from the newly isolated Penicillium crustosum FP 11 and its potential in the prebleaching of kraft pulp were evaluated using a statistical approach. A Plackett–Burman design (PBD) was carried out to select the significant variables of the medium, these being NaNO₃, KH₂PO₄, MgSO₄, KCl, Fe₂(SO₄)₃, yeast extract, corn stover, and initial pH, in a liquid culture under static conditions for 6 d at 28?°C. Statistical analysis with a central composite design and response surface methodology showed that 0.15% (w/v) KH₂PO₄, 2% (w/v) corn stover, and an initial pH of 6.0 provided the best conditions for xylanase production. Furthermore, xylanase from P. crustosum FP 11 was effective in the bleaching of Eucalyptus kraft pulp, with a significant kappa efficiency of 35.04%. Therefore, the newly isolated P. crustosum FP 11 from the Atlantic Forest biome in Brazil showed two advantages: xylanase production with agricultural residue (corn stover) as a carbon source and an improvement in the bleaching of kraft pulp. Environmental pollution could thus be minimized because of a reduction in the use of chlorine as a bleaching agent.     

Effects of curcumin on methyl methanesulfonate damage to mouse kidney

Methylmethane sulfonate (MMS) is an alkylating agent that may react with DNA and damage it. We investigated histological changes and apoptosis caused by MMS and the effects of curcumin on MMS treated mouse kidneys. Twenty-four mice were divided into four equal groups: controls injected with saline, a group injected with 40 mg/kg MMS, a group injected with 40 mg/kg MMS and given 100 mg/kg curcumin by gavage, and a group given 100 mg/kg curcumin by gavage. MMS caused congestion and vacuole formation, and elevated the apoptotic index significantly, but had no other effect on kidney tissue. Curcumin improved the congestion and vacuole formation caused by MMS and decreased the apoptotic index. Curcumin administered with MMS appears to decrease the deleterious effects of MMS on the kidney.     

Composition and Variation of the Human Milk Microbiota Are Influenced by Maternal and Early-Life Factors

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Culture of transgenic Drosophila melanogaster Schneider 2 cells in serum-free media based on TC100 basal medium

Requirements of eliminating animal proteins from cell culture have intensified in recent years, with the pressure of regulatory agencies related to biopharmaceuticals production. In this work, the substitution of fetal bovine serum by yeastolate and a soy hydrolysate (Hy Soy) for the culture of Drosophila melanogaster Schneider 2 cells transfected for the production of rabies virus G glycoprotein was evaluated. TC100 supplemented with glucose, glutamine, lipid emulsion and Pluronic F68 was employed as basal medium. Results show that yeastolate was more efficient on cell growth stimulation than Hy Soy. Cells adapted in medium formulation supplemented with 3 g/L yeastolate, 1% lipid emulsion, 10 g/L glucose, 3.5 g/L glutamine and 0.1% Pluronic F68 attained a maximum concentration of 10.7 x 10(6) cells/mL, with the expression of 9.4 ng/mL G glycoprotein.     

Studies on the cytotoxicity of miscellaneous compounds from Eupatorium betonicaeforme (D.C.) Baker (Asteraceae)

A detailed study on the cytotoxic effects of five known constituents isolated from the flowers and roots of Eupatorium betonicaeforme is reported, including 2,2-dimethyl-6-vinylchroman-4-one (1), 2-senecioyl-4-vinylphenol (2), 6-acetyl-2,2-dimethylchroman-4-one (3), (4E)-8beta-angeloyloxy-9beta,10beta-dihydroxy-1-oxogermacra-4,11(13)-dien-12,6alpha-olide (4), and 3beta-hydroxyicosan-1,5beta-olide (5). The sesquiterpene lactone 4 exhibited the highest cytotoxicity, with IC50 values ranging from 3.9 to 9.9 microM, showing some degree of cell selectivity. The antiproliferative activity of 4 was examined towards HL-60 cells, and found to diminish cell viability in a dose-dependent manner. Moreover, at all concentrations tested, there was a decrease in the number of cells capable of incorporating 5-bromo-2'-deoxyuridine (BrdU), indicating disruption of DNA synthesis. The morphological changes induced by 4 were compatible with apoptotic cell death. This work, thus, corroborates the anticancer potential of Eupatorium secondary metabolites.