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.     

Comparison of VHH‐Fc antibody production in Arabidopsis thaliana,Nicotiana benthamiana and Pichia pastoris

VHHs or nanobodies are widely acknowledged as interesting diagnostic and therapeutic tools. However, for some applications, multivalent antibody formats, such as the dimeric VHH‐Fc format, are desired to increase the functional affinity. The scope of this study was to compare transient expression of diagnostic VHH‐Fc antibodies in Nicotiana benthamiana leaves with their stable expression in Arabidopsis thaliana seeds and Pichia pastoris. To this end, VHH‐Fc antibodies targeting green fluorescent protein or the A. thaliana seed storage proteins (albumin and globulin) were produced in the three platforms. Differences were mainly observed in the accumulation levels and glycosylation patterns. Interestingly, although in plants oligomannosidic N‐glycans were expected for KDEL‐tagged VHH‐Fcs, several VHH‐Fcs with an intact KDEL‐tag carried complex‐type N‐glycans, suggesting a dysfunctional retention in the endoplasmic reticulum. All VHH‐Fcs were equally functional across expression platforms and several outperformed their corresponding VHH in terms of sensitivity in ELISA.     

N端缺失突变对核糖核酸酶抑制因子活性的影响

人胎盘核糖核酸酶抑制因子(HRI)是一种存在于细胞浆中的50 kD的酸性蛋白质,富含亮氨酸和半肤氨酸.作为胞浆蛋白可保护细胞不受外来胰RN白s。侵袭.HRI主要结构是由7个富含亮氨酸的重复序列组成,7个亮氨酸重复单位有规律环状排列使N端、c端在空间上较为接近.用PcR方法在HRI cl〕NAS,端去除30个核普酸,并将此缺失突变的HRI的cl〕NA片段构建于质粒pPIcgK,电击转化入毕赤酵母(Pi峨i。 Pasto汀S)Gslls中,进行分泌型表达.对表达产物进行亲和层析纯化.实验结果表明,N端缺失突变的HRI与RN白s。A的亲合力较野生型HRI降低1倍,但依然具有竞争性抑制RNas。A的活性,表明HRIN端10个氨基酸残基缺失后并未丧失其抑制活性.     

Functional importance of the family 1 glucosyltransferase UGT72B1 in the metabolism of xenobiotics in Arabidopsis thaliana

The Arabidopsis type 1 UDP-glucose-dependent glucosyltransferase UGT72B1 is highly active in conjugating the persistent pollutants 3,4-dichloroaniline (DCA) and 2,4,5-trichlorophenol (TCP). To determine its importance in detoxifying xenobiotics in planta, mutant plants where the respective gene has been disrupted by T-DNA insertion have been characterized. Extracts from the knockout ugt72B1 plants showed radically reduced conjugating activity towards DCA and TCP and the absence of immunodetectable UGT72B1 protein. In contrast, activities towards phenolic natural products were unaffected. When aseptic root cultures were fed [14C]-DCA, compared with wild types, the ugt72B1 plants showed a reduced rate of uptake of the xenobiotic and very little metabolism to soluble DCA-glucose or associated polar conjugates. Instead, the knockouts accumulated non-extractable radioactive residues, most probably associated with lignification. When the feeding studies were carried out with [14C]-TCP, rates and routes of metabolism were identical in the wild type and knockouts, with TCP-glucoside a major product in both cases. Similar differential effects on the metabolism of DCA and TCP were obtained in whole plant studies with wild type and ugt72B1 mutants, demonstrating that while UGT72B1 had a central role in metabolizing chloroanilines in Arabidopsis, additional UGTs could compensate for the conjugation of TCP in the knockout. TCP was equally toxic to wild type and ugt72B1 plants, while surprisingly, the knockouts were less sensitive to DCA. From this it was concluded that the glucosylation of DCA may not be as effective in xenobiotic detoxification as bound-residue formation.     

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.     

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.     

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.     

构巢曲霉内切纤维素酶A10在毕赤酵母中的表达及重组蛋白质的表征

内切纤维素酶是纤维素酶高效降解纤维素的一个关键因子,已广泛应用于工业生物技术领域。对来源于腐生真菌构巢曲霉的一个内切纤维素酶进行过表达及详细的酶学性质研究,研究结果表明:该内切纤维素酶在摇瓶和发酵罐条件下都成功获得表达,发酵罐条件下的蛋白质表达量达到0.89 mg/mL;该酶的最适反应p H和温度分别为4.0和80℃,在pH 2.0–12.0之间表现出了很好的稳定性;在温度￡60℃时,该酶非常稳定,当温度370℃,酶的稳定性大大降低;Co~(2+)、Mn~(2+)和Fe~(2+)促进了CMCase活性,而Pb~(2+)、Ni~(2+)和Cu~(2+)等金属离子表现出了一定的抑制作用。因此,该构巢曲霉内切纤维素酶表现出了非常好的耐酸、耐碱以及一定的耐热性等性能,具有开发为商品酶的潜力,为深入开发构巢曲霉来源糖苷酶的应用奠定了基础。     

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.     

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.     

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.     

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.