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| 2. |
- Collén, Anna, et al.
(författare)
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Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems - Effect of fusion localization on endoglucanase I of Trichoderma reesei
- 2001
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 910:2, s. 275-284
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Tidskriftsartikel (refereegranskat)abstract
- Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.
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| 3. |
- Collén, Anna
(författare)
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Hydrophobic Fusion Tags: Implications For Bioseparation and Cellular Expression
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The studies in this thesis have shown that the partitioning of endoglucanase I (EGI, Cel7B) from T. reesei could be significantly improved by relatively minor genetic engineering. By adding short peptides composed of tryptophan and proline residues to EGI, extreme partitioning could be obtained. The site of the tag fusion was shown to be crucial for the efficiency of the tag. Methods suitable for large-scale purification of genetically modified EGI by a single-step extraction in aqueous two-phase systems have been established. The most optimal fusion protein, with respect to partitioning enhancement resulted, however, in impaired production in T. reesei. This was further elucidated and the low production was suggested to be caused by several factors such as proteolysis, impaired secretion and possible down-regulation of the promoter due to intracellular accumulation of the hydrophobic fusion protein. At certain stages during growth of the transformant expressing EGIcore-P5(WP)4 slight induction of the gene encoding the ER residual chaperone BIPI was detected. Furthermore, the amphiphilic protein hydrophobin I was utilized as a fusion tag to direct partitioning in aqueous two-phase systems. A system with improved separation features was evaluated, which is a clear progression from previously used systems both with respect to both robustness and purification properties. Applications towards large-scale purification with this system might be possible in the foreseeable future. Additionally, a novel approach for detergent removal after two-phase extraction in detergent based systems was developed. By addition of thermoseparating polymers, HM-EOPO or EOPO, phase separation could be induced by temperature increase, and thus the fusion protein could be recovered in a water phase. This method is both environmentally benign and displays compatibility with subsequent purification steps and handling of the target protein.
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| 4. |
- Collén, Anna, et al.
(författare)
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Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems.
- 2002
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 78:4, s. 385-394
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Tidskriftsartikel (refereegranskat)abstract
- Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.
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| 5. |
- Collén, Anna, et al.
(författare)
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Protein production and induction of the unfolded protein response in Trichoderma reesei strain rut-c30 and its transformant expressing endoglucanase I with a hydrophobic tag
- 2005
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 89:3, s. 335-344
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Tidskriftsartikel (refereegranskat)abstract
- The effect of induction of protein production was studied in bioreactor cultures of T. reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag. The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production. Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations. Induction of the cellulase genes followed a similar temporal pattern in both strains, The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed. Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter. The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein. The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions. However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain.
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| 6. |
- Collen, A, et al.
(författare)
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VEGFA mRNA for regenerative treatment of heart failure
- 2022
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Ingår i: Nature reviews. Drug discovery. - : Springer Science and Business Media LLC. - 1474-1784 .- 1474-1776. ; 21:1, s. 79-80
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 7. |
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| 8. |
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| 9. |
- Collen, J, et al.
(författare)
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Farming and physiology of the red algae Eucheuma: Growing commercial importance in East Africa
- 1995
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Ingår i: AMBIO. - : ROYAL SWEDISH ACAD SCIENCES. - 0044-7447. ; 24:7-8, s. 497-501
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Red marine macroalgae of the genus Eucheuma were introduced from the Philippines to Zanzibar in 1989. Their cultivation is of growing importance, especially on the islands of Zanzibar and Pemba. The algae are cultivated for the production of carrageenan,
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| 10. |
- COLLEN, J, et al.
(författare)
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PHOTOSYNTHETIC PRODUCTION OF HYDROGEN-PEROXIDE BY ULVA-RIGIDA C-AG (CHLOROPHYTA)
- 1995
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Ingår i: PLANTA. - : SPRINGER VERLAG. - 0032-0935. ; 196:2, s. 225-230
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Production of hydrogen peroxide has been found in Ulva rigida (Chlorophyta). The formation of H2O2 was light dependent with a production of 1.2 mu mol . g FW-1. h(-1) in sea water (pH 8.2) at an irradiance of 700 mu mol photons m(-2). s(-1). The excretion
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