Expression of the Arabidopsis thaliana AtJ2 cochaperone protein in Pichia pastoris

A vector was constructed for intracellular expression of the Arabidopsis thaliana DnaJ homologue AtJ2 in the methylotrophic yeast Pichia pastoris. The vector includes DNA encoding an amino-terminal histidine-tag, to simplify protein purification. Shake-flask cultures could be induced to produce approximately 250 mg/ L of AtJ2. Purified recombinant AtJ2 was able to stimulate the ATPase activities of both the Escherichia coli and Zea mays cytoplasmic Stress70 chaperone proteins five- to ninefold. The carboxy terminus of AtJ2 is -CAQQ, a protein farnesylation motif. When transformed P. pastoris was induced to synthesize AtJ2 in the presence of [(3)H]mevalonolactone, radioactivity was incorporated into the protein, suggesting farnesylation.     

Overexpression of Arabidopsis thaliana soluble epoxide hydrolase 1 in Pichia pastoris and characterisation of the recombinant enzyme

Epoxide hydrolases are enzymes involved in metabolism and defense of plants. Genome scanning suggested the presence of several genes encoding epoxide hydrolase in Arabidopsis thaliana. To assure that the predicted genes are functional and the translated products have epoxide hydrolase activity analysis at the protein level is needed. We have started to clone the cDNAs and overexpress them for catalytic and physico-chemical analysis. We here report that Pichia pastoris serves as an efficient system for overexpression of soluble epoxide hydrolase 1 (AtsEH1) from A. thaliana. A tag containing six histidine residues was added to the N-terminus to enable efficient one-step purification on nickel-agarose. The enzyme was expressed at levels >18 mg.L(-1) of culture and a French Press was found to be effective to achieve cell lysis. The recombinant enzyme had a molecular mass of 37 or 38 kDa based on SDS-PAGE or MALDI-TOF analysis, respectively. The enzyme was highly active towards the substrate trans-stilbene oxide (TSO) and had a pH optimum at 7 and a temperature optimum at 54 degrees C. Using TSO as substrate the K(m) and V(max) values were determined to 5 micro M and 2 micromol min(-1) mg protein(-1), respectively. The activity was 50-fold lower towards cis-stilbene oxide. The stability over time was tested from 20 to 54 degrees C and the enzyme lost activity at varying degrees at the temperatures tested but was stable for several months at 4 degrees C.     

The Role of Water Channel Proteins in Facilitating Recovery of Leaf Hydraulic Conductance from Water Stress in Populus trichocarpa

Gas exchange is constrained by the whole-plant hydraulic conductance (K _plant). Leaves account for an important fraction of K _plant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (K _leaf) decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs). Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, K _leaf recovered only 2 hours after plants were rewatered. Recovery of K _leaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs) showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in K _leaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in K _leaf.     

Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris

Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris , which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae . Using P. pastoris psd1 Δ and psd2 Δ mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP)–ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris , which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes.     

Different Phototransduction Kinetics of Phytochrome A and Phytochrome B in Arabidopsis thaliana

The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of β-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h⁻¹ (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h⁻¹ caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b₅₆₁ proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b₅₆₁-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b₅₆₁ paralogs from Arabidopsis thaliana (Acytb₅₆₁-A, Acytb₅₆₁-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb₅₆₁-A resembles the best characterised member of the CYBASC family, the cytochrome b₅₆₁ from adrenomedullary chromaffin vesicles, and that Acytb₅₆₁-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (E_M) values were found to be fully consistent with ascorbate oxidation activities and Fe^3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b₅₆₁ from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E_M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E_M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe^3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E_M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b₅₆₁ paralogs exist as homodimers.     

Purification and characterization of inulinase from Aspergillus niger AF10 expressed in Pichia pastoris

The inuA1 gene encoding an exoinulinase from Aspergillus niger AF10 was expressed in Pichia pastoris, and the recombinant enzyme activity was 316U/ml in a 5L fermentor, with the inulinase protein accounting for 35% of the total protein of fermentation broth. The hydrolysis rate of inulin can reach 92%, with a 25U/g inulin enzyme addition, and 90% of fructose content after 6h. Glucose can significantly inhibit the enzymatic hydrolysis of inulin. This is the first report of glucose inhibition of inulinase-catalyzed hydrolysis.     

Higher Activity of an Aldehyde Oxidase in the Auxin-Overproducing superroot1 Mutant of Arabidopsis thaliana

Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1–3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b(561) enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers.     

Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in Φ_PSII and q _L in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.     

Regulation of phytochrome B signaling by phytochrome A and FHY1 in Arabidopsis thaliana

Phytochrome A (phyA) and phytochrome B (phyB) share the control of many processes but little is known about mutual signaling regulation. Here, we report on the interactions between phyA and phyB in the control of the activity of an Lhcb1*2 gene fused to a reporter, hypocotyl growth and cotyledon unfolding in etiolated Arabidopsis thaliana. The very-low fluence responses (VLFR) induced by pulsed far-red light and the high-irradiance responses (HIR) observed under continuous far-red light were absent in the phyA and phyA phyB mutants, normal in the phyB mutant, and reduced in the fhy1 mutant that is defective in phyA signaling. VLFR were also impaired in Columbia compared to Landsberg erecta. The low-fluence responses (LFR) induced by red-light pulses and reversed by subsequent far-red light pulses were small in the wild type, absent in phyB and phyA phyB mutants but strong in the phyA and fhy1 mutants. This indicates a negative effect of phyA and FHY1 on phyB-mediated responses. However, a pre-treatment with continuous far-red light enhanced the LFR induced by a subsequent red-light pulse. This enhancement was absent in phyA, phyB, or phyA phyB and partial in fhy1. The levels of phyB were not affected by the phyA or fhy1 mutations or by far-red light pre-treatments. We conclude that phyA acting in the VLFR mode (i.e. under light pulses) is antagonistic to phyB signaling whereas phyA acting in the HIR mode (i.e. under continuous far-red light) operates synergistically with phyB signaling, and that both types of interaction require FHY1.     

Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

Background

Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.

Methodology/Principal Findings

We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.

Conclusions/Significance

The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.     

Molecular cloning and characterization of the Arabidopsis thaliana alpha-subunit of elongation factor 1B

Using a PCR-based approach, we have isolated two Arabidopsis thaliana cDNA clones (alpha1 and alpha2) encoding the alpha-subunit of translation elongation factor 1B (eEF1Balpha). They encode open reading frames of 228 and 224 amino acids respectively, with extensive homology to eEF1Balpha subunits from different organisms, particularly in the C-terminal half of the protein. They both lack a conserved phosphorylation site that has been implicated in regulating nucleotide exchange activity. Using a plasmid shuffling experiment, we demonstrated that both alpha1 and alpha2 clones are able to complement a mutant yeast strain deficient for the eEF1Balpha subunit. This provides evidence that Arabidopsis encodes at least two functional isoforms of this subunit, termed eEF1Balpha1 and eEF1Balpha2. A third cDNA clone was isolated that appeared to result from an alternative splicing event of the eEF1Balpha1 gene.     

Metabolism of Zearalenone by Genetically Modified Organisms Expressing the Detoxification Gene from Clonostachys rosea

Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101. An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli. Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized. Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45°C and pH 10.5 (k_cat at 30°C, 0.51 s⁻¹). The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors. ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the k_cat was highest with zearalanone (1.5 s⁻¹) and lowest with β-zearalenol (0.075 s⁻¹). EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity. Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions. We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN. While recombinant E. coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, β-zearalenol, by an as-yet-unidentified reductase and remained in the medium. Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H. El-Nezami, N. Polychronaki, S. Salminen, and H. Mykkuäne, Appl. Environ. Microbiol. 68:3545-3549, 2002). An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.     

毕赤酵母表达的重组乙肝表面抗原SS1的纯化及性质鉴定

对毕赤酵母（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）表达的重组乙肝表面抗原ＳＳ１的纯化以及蛋白质的理化性质及免疫原性进行了进一步的研究。采用单抗亲和层析的方法,一步纯化即可得到纯度为９５％的纯化蛋白质。ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ印迹鉴定表明,纯化的ＳＳ１融合蛋白同时具有良好的Ｓ和ＰｒｃＳ１抗原性。ＣｓＣ１密度梯度离心和电镜检测的结果则表明,这一重组抗原可以形成与ＨＢＶ亚病毒颗粒类似的颗粒结构。在ＢＡＬＢ／