| 1. |
- Arnell, Robert, et al.
(författare)
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Biotechnological Approach to the Synthesis of 9α-Hydroxylated Steroids
- 2007
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Ingår i: Preparative Biochemistry & Biotechnology. - : Informa UK Limited. - 1082-6068 .- 1532-2297. ; 37:4, s. 309-321
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Tidskriftsartikel (refereegranskat)abstract
- The steroid 9α-hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9α-hydroxyprogesterone. In 7 days, 43.6 mg9α-hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4-androstene-3,17-dione in vivo. An extensive analytical method development, including LLE, HPLC-DAD, MS, andNMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion ratewere unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.
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| 2. |
- Heldtander Königsson, Malin, et al.
(författare)
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Detection of Renibacterium salmoninarum in tissue samples by sequence capture and fluorescent PCR based on the 16S rRNA gene
- 2005
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Ingår i: Veterinary Microbiology. ; 105:3-4, s. 235-243
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Tidskriftsartikel (refereegranskat)abstract
- The 16S rRNA genes from eight isolates of Renibacterium salmoninarum with different origins and dates of isolation weresequenced to evaluate the possibility to construct a diagnostic PCR system with target sites within this gene. The sequences werefound to be identical but for one single position in one of the isolates, and two regions with an adequate number of nucleotidedifferences as compared to closely related species were identified. Species-specific fluorescent PCR primers complementary tothese regions were constructed as well as oligonucleotides for DNA preparation by sequence capture. A mimic molecule wasconstructed to be used as an internal control. The PCR was specific and allowed the detection of DNA equivalent to 1–10 R.salmoninarum genomes per reaction. The DNA preparation with sequence capture and analysis by PCR with a mimic was foundto be a reliable method for analysis of kidneys from fish with BKD. The amount of PCR inhibiting substances present in thetissue was reduced, and the relevant DNAwas concentrated in the capture step. Furthermore, the use of the mimic molecule inthe system assured that false negative results could be identified.
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| 3. |
- Li, Jing-Jing, et al.
(författare)
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Immobilized Triton X-100-assisted refolding of Green Fluorescent Protein-Tobacco Etch Virus protease fusion protein using β-cyclodextrin as the eluent
- 2009
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Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113 .- 1873-3298. ; 44:3, s. 277-282
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Tidskriftsartikel (refereegranskat)abstract
- A new protein refolding technique based on the use of the non-charged detergent Triton X-100 immobilized to the cross-linked agarose gel Sepharose High Performance has been developed. The new solid phase was used in combination with soluble β-cyclodextrin (β-CD) to refold recombinant Green Fluorescent Protein fused to Tobacco Etch Virus protease (GFPTEVP) expressed as inclusion bodies in E. coli. Previous attempts to refold recombinant GFPTEVP by dilution had failed. In the new procedure a column packed with Triton X-100-coupled Sepharose High Performance was used to capture unfolded GFPTEVP followed by elution using an increasing β-CD concentration gradient. The yield of properly refolded GFPTEVP was 46% at a protein concentration of 380 μg/ml. In contrast, dilution refolding of GFPTEVP at 200 μg/ml refolding buffer resulted in only 4.7% of native protein.
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| 4. |
- Luo, J, et al.
(författare)
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Size characterization of green fluorescent protein inclusion bodies in E. coli using asymmetrical flow field-flow fractionation-multi-angle light scattering
- 2006
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1120:1-2, s. 158-164
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Konferensbidrag (refereegranskat)abstract
- The goal of this study was to investigate the applicability of asymmetrical flow field-flow fractionation-multi-angle light scattering (AsFIFFFMALS) for size analysis of green fluorescent protein inclusion bodies (GFPIBs). The size distributions of GFPIBs prepared by various culture conditions were determined. For GFPIBs prepared at 37 degrees C the peak maximum hydrodynamic diameter (dH) first increased and then decreased with the increase of the induction times in the presence of 0.1 and 2 mM isopropyl-beta-D-thiogalactoside (IPTG). For GFPIBs prepared at 30 degrees C the peak maximum dH was constant at about 700 nm irrespectively of the induction times and IPTG concentrations.
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| 5. |
- Macvanin, Mirjana, et al.
(författare)
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Fusidic acid-resistant mutants of Salmonella enterica serovar typhimurium have low levels of heme and a reduced rate of respiration and are sensitive to oxidative stress
- 2004
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 48:10, s. 3877-3883
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in the translation elongation factor G (EF-G) make Salmonella enterica serovar Typhimurium resistant to the antibiotic fusidic acid. Fusr mutants are hypersensitive to oxidative stress and rapidly lose viability in the presence of hydrogen peroxide. We show that this phenotype is associated with reduced activity of two catalase enzymes, HPI (a bifunctional catalase-hydroperoxidase) and HPII (a monofunctional catalase). These catalases require the iron-binding cofactor heme for their activity. Fusr mutants have a reduced rate of transcription of hemA, a gene whose product catalyzes the first committed step in heme biosynthesis. Hypersensitivity of Fusr mutants to hydrogen peroxide is abolished by the presence of -aminolevulinic acid, the precursor of heme synthesis, in the growth media and by the addition of glutamate or glutamine, amino acids required for the first step in heme biosynthesis. Fluorescence measurements show that the level of heme in a Fusr mutant is significantly lower than it is in the wild type. Heme is also an essential cofactor of cytochromes in the electron transport chain of respiration. We found that the rate of respiration is reduced significantly in Fusr mutants. Sequestration of divalent iron in the growth media decreases the sensitivity of Fusr mutants to oxidative stress. Taken together, these results suggest that Fusr mutants are hypersensitive to oxidative stress because their low levels of heme reduce both catalase activity and respiration capacity. The sensitivity of Fusr mutants to oxidative stress could be associated with loss of viability due to iron-mediated DNA damage in the presence of hydrogen peroxide. We argue that understanding the specific nature of antibiotic resistance fitness costs in different environments may be a generally useful approach in identifying physiological processes that could serve as novel targets for antimicrobial agents.
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| 6. |
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