An antioxidant protein in Curcuma comosa Roxb. Rhizomes

Curcuma comosa Roxb. is an indigenous Thai herb which is usually used as a food ingredient but it is also used in traditional folk medicine for the treatment of uterine inflammation. The crude protein extract from the rhizomes of this plant was found to possess free radical scavenging capacity, as detected by the DPPH assay. This antioxidant activity was purified by DEAE anion exchange chromatography to a fraction (called IE-1) that was comprised of a single main protein band of ∼18 kDa (UB-DEAE), as determined by SDS–PAGE resolution with Coomassie blue staining, and had a specific activity of 193.8 U/mg. In-gel trypsin digestion of the SDS–PAGE resolved UB-DEAE band followed by liquid chromatography–tandem mass spectrometry produced three reliably sequenced peptides, which all were found to very likely be fragments of a superoxide dismutase homologue (SOD, EC 1.15.1.1), an antioxidant enzyme that has been found in several plants. In support of this notion, the IE-1 fraction was found to yield positive results with the riboflavin–nitroblue tetrazolium (NBT) assay, a standard test for SOD-like activity. Together, these data then support the existence of an SOD homologue antioxidant protein in the rhizomes of C. comosa as a contributing agent to the total observed antioxidant activity.     

Isolation and characterization of the carbon catabolite‐derepressing protein kinase Snf1 from the stress tolerant yeast Torulaspora delbrueckii

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae snf1Δ mutant strain. DNA sequence analysis revealed that the fragment contained a complete open reading frame (ORF), which shares a high similarity with the S. cerevisiae energy sensor protein kinase Snf1. The cloned TdSNF1 gene was able to restore growth of the S. cerevisiae snf1Δ mutant strain on media containing nonfermentable carbon sources. Furthermore, cells of the Tdsnf1Δ mutant were unable to proliferate under nonfermenting conditions. Finally, protein domain analysis showed that TdSnf1p contains a typical catalytic protein kinase domain (positions 41–293), which is also present in other Snf1p homologues. Within this region we identified a protein kinase ATP‐binding region (positions 48–71) and a consensus Ser/Thr protein kinase active site (positions 160–172). The GenBank Accession No. for the sequenced DNA fragment is HM131845. Copyright © 2010 John Wiley & Sons, Ltd.     

Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae

Fluorescent proteins are convenient tools for measuring protein expression levels in the budding yeast Saccharomyces cerevisiae. Co‐expression of proteins from distinct vectors has been seen by fluorescence microscopy; however, the expression of two fluorescent proteins on the same vector would allow for monitoring of linked events. We engineered constructs to allow dicistronic expression of red and green fluorescent proteins and found that expression levels of the proteins correlated with their order in the DNA sequence, with the protein encoded by the 5′‐gene more highly expressed. To increase expression levels of the second gene, we tested four regulatory elements inserted between the two genes: the IRES sequences for the YAP1 and p150 genes, and the promoters for the TEF1 gene from both S. cerevisiae and Ashbya gossypii. We generated constructs encoding the truncated ADH1 promoter driving expression of the red protein, yeast‐enhanced Cherry, followed by a regulatory element driving expression of the green protein, yeast‐enhanced GFP. Three of the four regulatory elements successfully enhanced expression of the second gene in our dicistronic construct. We have developed a method to express two genes simultaneously from one vector. Both genes are codon‐optimized to produce high protein levels in yeast, and the protein products can be visualized by microscopy or flow cytometry. With this method of regulation, the two genes can be driven in a dicistronic manner, with one protein marking cells harbouring the vector and the other protein free to mark any event of interest. Copyright © 2009 John Wiley & Sons, Ltd.     

Improvement of functional properties of acid-precipitated soy protein by the attachment of dextran through Maillard reaction

The functional acid-precipitated soy protein (SAPP)–dextran conjugate was prepared by dry-heated storage at 60 °C under 79% relative humidity (RH) for 5 days through Maillard reaction between the ε-amino of lysine in soy proteins and the reducing-end carbonyl residue in the dextran. The covalent attachment of dextran to SAPP was confirmed by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography. Functional properties of soy protein depend on the structural and aggregation characteristics of their major components (storage globulins 7S and 11S). The conjugate seemed to be predominantly formed by 7S, and the acidic subunits of 11S in soy protein. The emulsifying properties of the SAPP–dextran conjugate were about four times higher than those of SAPP. The solubility of the protein was not enhanced as a result of preheating, but rather it was not decreased when the conjugated protein was heated at 90 °C for 20 min due to the presence of the polysaccharide. The excellent emulsifying properties of SAPP–dextran conjugate were maintained even at pH 3.0 and were further improved at pH 10.0. The object of Maillard reaction is to guarantee the suitable reaction degree, and the resulting soluble conjugate can have excellent emulsifying properties.     

A proteomic analysis of green and white sturgeon larvae exposed to heat stress and selenium

Temperature and selenium are two environmental parameters that potentially affect reproduction and stock recruitment of sturgeon in the San Francisco Bay/Delta Estuary. To identify proteins whose expression is modified by these environmental stressors, we performed a proteomic analysis on larval green and white sturgeons exposed to 18 or 26 °C and micro-injected with Seleno-L-Methionine to reach 8 µg g⁻¹ selenium body burden, with L-Methionine as a control. Selenium and high temperature induced mortalities and abnormal morphologies in both species, with a higher mortality in green sturgeon. Larval proteins were separated by two-dimensional gel electrophoresis and differential abundances were detected following spot quantitation and hierarchical cluster analysis. In green sturgeon, 34 of 551 protein spots detected on gels showed a variation in abundance whereas in white sturgeon only 9 of 580 protein spots were differentially expressed (P < 0.01). Gel replicates were first grouped according to heat treatment. Fifteen of these spots were identified using MALDI TOF/TOF mass spectrometry. Proteins involved in protein folding, protein synthesis, protein degradation, ATP supply and structural proteins changed in abundance in response to heat and/or selenium. 40S ribosomal protein SA, FK506-binding protein 10, 65 kDa regulatory subunit A of protein phosphatase 2, protein disulfide isomerase, stress-induced-phosphoprotein 1, suppression of tumorigenicity 13 and collagen type II alpha 1, were differentially expressed in high temperature treatment only. Serine/arginine repetitive matrix protein 1, creatine kinase, serine peptidase inhibitor Kazal type 5 and HSP90 were sensitive to combined temperature and selenium exposure. Valosin-containing protein, a protein involved in aggresome formation and in protein quality control decreased more than 50% in response to selenium treatment. Potential use of such proteins as biomarkers of environmental stressors in larval sturgeons could indicate early warning signals preceding population decline.