| 1. |
- Beaujean, A, et al.
(författare)
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Engineering direct fructose production in processed potato tubers by expressing a bifunctional alpha-amylase/glucose isomerase gene complex
- 2000
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Ingår i: Biotechnology and Bioengineering. - 0006-3592. ; 70:1, s. 9-16
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Tidskriftsartikel (refereegranskat)abstract
- Manipulation of starch biosynthesis/degradation and formation of novel molecules in storage organs of plants through genetic engineering is an attractive but technically challenging goal. We report here, for the first time, that starch was degraded and glucose and fructose were produced directly when crushed potato tubers expressing a starch degrading bifunctional gene were heated for 45 minutes at 65 degrees C. To achieve this, we have constructed a fusion gene encoding the thermostable enzymes: alpha-amylase (Bacillus stearothermophilus) and glucose isomerase (Thermus thermophilus). The chimeric gene was placed under the control of the granule-bound-starch synthase promoter. This enzymatic complex produced in transgenic tubers was only active at high temperature (65 degrees C). More than 100 independent transgenic potato plants were regenerated. Molecular analyses confirmed the stable integration of the chimeric gene into the potato genome. The biochemical analyses performed on young and old tubers after high-temperature treatment (65 degrees C) revealed an increase in the formation rate of fructose and glucose by a factor of 16.4 and 5. 7, respectively, in the transgenic tubers as compared to untransformed control tubers. No adverse discernible effect on plant development and metabolism including tuber formation and starch accumulation was observed in the transgenic plants before heat treatment. Our results demonstrate that it is possible to replace starch degradation using microbial enzymes via a system where the enzymes are produced directly in the plants, but active only at high temperature, thus offering novel and viable strategies for starch-processing industries.
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| 2. |
- Carlsson, Magnus L.R., et al.
(författare)
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Expression, Purification and Initial Characterization of Functional α1-Microglobulin (A1M) in Nicotiana benthamiana
- 2020
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- α1-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.
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