| 1. |
- Gronowitz, J S, et al.
(author)
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Carrier bound templates for single tube reverse transcriptase assays and for combined purification and activity analyses, with special reference to HIV
- 1991
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In: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 13:1, s. 127-142
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Journal article (peer-reviewed)abstract
- Polyriboadenosine (prA) was coupled to polycarbonate macrobeads or magnetic beads. The efficiency of the beads and of prA-Sepharose, after priming with odT, as templates in activity assays of purified AMV- and HIV-reverse transcriptase (RT), using [125I]iododeoxyuridine-triphosphate as substrate, was studied. Although the use of immobilized templates, compared with soluble template, resulted in a decreased total molar turnover, it did not affect the sensitivity of the assay for detecting RT. The utility of the new assay was analyzed by mixing purified AMV- or HIV-Rt with different dilutions of the untreated clinical specimen. This showed that RT activity was unaffected by 100 microliters of an extract of whole blood cells resuspended to their original blood volume and diluted 1/64, and also by 100 microliters of serum diluted 1/64. To improve the utility of the assay at the inhibitory concentrations of clinical specimens, the following procedure was adopted: the sample to be analyzed was incubated with the carrier bound template in order to allow the RT to bind, the carrier was washed to remove inhibitory factors, and the reaction components were then added to determine the amount of bound RT. This procedure greatly enhanced the recovery of RT activity from crude specimens and made the direct detection of HIV-RT possible. The assay is easily automated and useful for RT determination in multiple samples and for determining RT-inhibiting substances such as substrate analogs and antibodies.
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| 2. |
- Andersson, C., et al.
(author)
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Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column
- 2001
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In: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 34, s. 25-32
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Journal article (peer-reviewed)abstract
- The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.
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| 3. |
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| 4. |
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| 5. |
- Blomqvist, Johanna, et al.
(author)
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Temperature-dependent changes in the microbial storage flora of birch and spruce sawdust
- 2014
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In: Biotechnology and Applied Biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 61, s. 58-64
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Journal article (peer-reviewed)abstract
- Sawdust can be used to make pellets (biofuel) and particle boards and as a potential lignocellulose feedstock in bioethanol production. Microbial activity can affect sawdust quality; hence, we monitored the microbial population in birch- and spruce sawdust after 3 months' storage at various temperatures. Species composition was similar on both materials but was strongly influenced by temperature. Bacteria were present on all materials at all conditions: on birch, 2.8x10(8), 1.1x10(8), and 8.8x10(6), and on spruce, 4.1x10(8), 5.6x10(7), and 1.5x10(8)CFU/g DM, at 2, 20, and 37 degrees C, respectively. Dominant bacteria at 2, 20, and 37 degrees C were Pseudomonas spp. (some Enterobacteriaceae spp. present), Luteibacter rhizovicinus, and Fulvimonas sp., respectively. Pseudomonas spp. were absent at 20 degrees C. Among microfungi, yeasts dominated at 2 degrees C but were absent at 37 degrees C, whereas molds dominated at 20 and 37 degrees C. Common yeasts included Cystofilobasidium capitatum, Cystofilobasidium infirmominiatum, Candida saitoana, Candida oregonensis, and Candida railenensis. Ophiostoma quercus was a common mold at 2 and 20 degrees C, whereas the human pathogens Aspergillus fumigatus and Paecilomyces variotii dominated at 37 degrees C. Attempts to influence the microflora by addition of the biocontrol yeasts, Wickerhamomyces anomalus and Scheffersomyces stipitis, were unsuccessful, as their growth in sawdust was poor to absent.
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| 6. |
- Chandolias, Konstantinos, et al.
(author)
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Protective effect of a reverse membrane bioreactor against toluene and naphthalene in anaerobic digestion
- 2022
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In: Biotechnology and Applied Biochemistry. - : Wiley. - 1470-8744 .- 0885-4513. ; 69:3, s. 1267 -1274
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Journal article (peer-reviewed)abstract
- Raw syngas contains tar contaminants including toluene and naphthalene, which inhibit its conversion to methane. Cell encasement in a hydrophilic reverse membrane bioreactor (RMBR) could protect the cells from hydrophobic contaminants. This study aimed to investigate the inhibition of toluene and naphthalene and the effect of using RMBR. In this work, toluene and naphthalene were added at concentrations of 0.5–1.0 and 0.1–0.2 g/L in batch operation. In continuous operation, concentration of 0–6.44 g/L for toluene and 0–1.28 g/L for naphthalene were studied. The results showed that no inhibition was observed in batch operation for toluene and naphthalene at concentrations up to 1 and 0.2 g/L, respectively. In continuous operation of free cell bioreactors (FCBRs), inhibition of toluene and naphthalene started at 2.05 and 0.63 g/L, respectively. When they were present simultaneously, inhibition of toluene and naphthalene occurred at concentrations of 3.14 and 0.63 g/L, respectively. In continuous RMBRs, no inhibition for toluene and less inhibition for naphthalene were observed, resulting in higher methane production from RMBR than that of FCBR. These results indicated that RMBR system gave a better protection effect against inhibitors compared with FCBR.
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| 7. |
- Eriksson, Cecilia, et al.
(author)
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Affibody molecule-mediated depletion of HSA and IgG using different buffer compositions : a 15 min protocol for parallel processing of 1-48 samples
- 2010
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In: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 56, s. 49-57
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Journal article (peer-reviewed)abstract
- High-abundant plasma proteins pose a challenge in a large number of proteomics-based technologies. Depletion of these high-abundant proteins has proven to be a fruitful strategy to circumvent masking of lower-abundant proteins that could serve as valuable biomarker candidates. However, current strategies often do not meet the throughput requirements of large-scale proteomic studies. In the present paper, a flexible and parallelized method for the depletion of high-abundant proteins is described, allowing the removal of the two most abundant proteins from 48 blood-derived samples in less than 15 min using Affibody molecules as affinity ligands. A sample-processing platform like this should be suitable for a number of proteomics technologies; its flexibility in buffer composition allows for different types of downstream applications.
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| 8. |
- Falk, Ronny, et al.
(author)
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A high-stringency proteomics concept aimed for generation of antibodies specific for cDNAencoded proteins
- 2002
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In: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 35:2, s. 75-82
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Journal article (peer-reviewed)abstract
- A novel dual bacterial expression system, designed for high-throughput generation of antibodies specific for cDNA-encoded proteins, is presented. The concept involves parallel expression of cDNA-encoded proteins, in two vector systems, as fusions with two different tags, both enabling single-step affinity purification under denaturing conditions. One of the fusion tags includes a portion with documented immunopotentiating effect to stimulate antibody production, and the generated fusion proteins are used to elicit antibodies. The second fusion protein is used in an immobilized form as an affinity ligand to enrich, from the generated antisera, antibodies with selective reactivity to the cDNA-encoded part. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The five antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the five antibodies gave specific staining in Western-blot screening of various cell types and tissue homogenates. When the same five cDNAs were processed and analysed using a single-vector method, antibodies with a more non-specific staining were generated. We thus conclude that the presented dual-vector method offers a highly stringent strategy for generation of monospecific polyclonal antibodies.
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| 9. |
- Falk, Ronny, et al.
(author)
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An improved dual-expression concept, generating high-quality antibodies for proteomics research
- 2003
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In: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 38, s. 231-239
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Journal article (peer-reviewed)abstract
- A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.
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| 10. |
- Friedman, Mikaela, et al.
(author)
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Engineered affinity proteins for tumour-targeting applications
- 2009
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In: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 53, s. 1-29
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Research review (peer-reviewed)abstract
- Targeting of tumour-associated antigens is an expanding treatment modality in clinical oncology as an alternative to, or in combination with, conventional treatments, such as chemotherapy, external-radiation therapy and surgery. Targeting of antigens that are unique or more highly expressed in tumours than in normal tissues can be used to increase the specificity and reduce the cytotoxic effect on normal tissues. Several targeting agents have been studied for clinical use, where monoclonal antibodies have been the ones most widely used. More than 20 monoclonal antibodies are approved for therapy today and the largest field is oncology. Advances in genetic engineering and in vitro selection technology has enabled the feasible high-throughput generation of monoclonal antibodies, antibody derivatives [e.g. scFvs, Fab molecules, dAbs (single-domain antibodies), diabodies and minibodies] and more recently also non-immunoglobulin scaffold proteins. Several of these affinity proteins have been investigated for both in vivo diagnostics and therapy. Affinity proteins in tumour-targeted therapy can affect tumour progression by altering signal transduction or by delivering a payload of toxin, drug or radionuclide. The ErbB receptor family has been extensively studied as biomarkers in tumour targeting, primarily for therapy using monoclonal antibodies. Two receptors in the ErbB family, EGFR (epidermal growth factor receptor) and HER2 (epidermal growth factor receptor 2), are over-expressed in various malignancies and associated with poor patient prognosis and are therefore interesting targets for solid turnours. In the present review, strategies are described for tumour targeting of solid turnours using affinity proteins to deliver radionuclides, either for molecular imaging or radiotherapy. Antibodies, antibody derivatives and non-immunoglobulin scaffold proteins are discussed with a certain focus on the affibody (Affibody (R)) molecule.
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