| 1. |
- Johansson, Tomas, et al.
(författare)
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Transcriptional responses of Paxillus involutus and Betula pendula during formation of ectomycorrhizal root tissue
- 2004
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Ingår i: Molecular Plant-Microbe Interactions. - 0894-0282 .- 1943-7706. ; 17:2, s. 202-215
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Tidskriftsartikel (refereegranskat)abstract
- In order to obtain information on genes specifically expressed in the ectomycorrhizal symbiosis, 3,555 expressed sequence tags (ESTs) were analyzed from a cDNA library constructed from ectomycorrhiza formed between the basidiomycete Paxillus involutus and birch (Betula pendula). cDNA libraries from saprophytically growing fungus (3,964 ESTs) and from axenic plants (2,532 ESTs) were analyzed in parallel. By clustering all the EST obtained, a nonredundant set of 2,284 unique transcripts of either fungal or plant origin were identified. The expression pattern of these genes was analyzed using cDNA microarrays. The analyses showed that the plant and fungus responded to the symbiosis by altering the expression levels of a number of enzymes involved in carbon metabolism. Several plant transcripts with sequence similarities to genes encoding enzymes in the tricarboxylic cycle and electron transport chain were down regulated as compared with the levels in free-living roots. In the fungal partner, a number of genes encoding enzymes in the lipid and secondary metabolism were down regulated in mycorrhiza as compared with the saprophytically growing mycelium. A substantial number of the ESTs analyzed displayed significant sequence similarities to proteins involved in biotic stress responses, but only a few of them showed differential expression in the mycorrhizal tissue, including plant and fungal metallothioneins and a plant defensin homologue. Several of the genes that were differentially expressed in the mycorrhizal root tissue displayed sequence similarity to genes that are known to regulate growth and development of plant roots and fungal hyphae, including transcription factors and Rho-like GTPases.
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| 2. |
- Gräslund, Torbjörn, et al.
(författare)
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Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology
- 2002
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 96:1, s. 93-102
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Tidskriftsartikel (refereegranskat)abstract
- The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.
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| 3. |
- Gräslund, Torbjörn, et al.
(författare)
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Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 942:1-2, s. 157-166
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Tidskriftsartikel (refereegranskat)abstract
- To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.
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| 4. |
- Hedhammar, My, et al.
(författare)
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Negatively charged purification tags for selective anion-exchange recovery
- 2004
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 17:11, s. 779-786
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Tidskriftsartikel (refereegranskat)abstract
- A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.
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| 5. |
- Hertzberg, M., et al.
(författare)
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A transcriptional roadmap to wood formation
- 2001
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 98:25, s. 14732-14737
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Tidskriftsartikel (refereegranskat)abstract
- The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.
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| 6. |
- Westberg, Joakim, et al.
(författare)
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The genome sequence of Mycoplasma mycoides subsp mycoides SC type strain PG1(T), the causative agent of contagious bovine pleuropneumonia (CBPP)
- 2004
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 14:2, s. 221-227
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Tidskriftsartikel (refereegranskat)abstract
- Mycoplasma mycoides subsp. mycoidesSC (MmymySC) is the etiological agent of contagious bovine pleuropneumonia (CBPP), a highly contagious respiratory disease in cattle. The genome of Mmymy SC type strain PUT has been sequenced to map all the genes and to facilitate further studies regarding the cell function of the organism and CBPP. The genome is characterized by a single circular chromosome of 1,211,703 bp with the lowest G+C content (24 mole%) and the highest density of insertion sequences (13% of the genome size) of all sequenced bacterial genomes. The genome contains 985 putative genes, of which 72 are part of insertion sequences and encode transposases. Anomalies in the GC-skew pattern and the presence of large repetitive sequences indicate a high genomic plasticity. A variety of potential virulence factors was identified, including genes encoding putative variable surface proteins and enzymes and transport proteins responsible for the production of hydrogen peroxide and the capsule, which is believed to have toxic effects on the animal.
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