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- Bandmann, Nina, 1971-
(author)
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Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification
- 2007
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Doctoral thesis (other academic/artistic)abstract
- The bacterium Escherichia coli (E. coli) is in many situations an ideal host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve sufficiently high product quantities. However, there are several factors that may limit this host’s ability to produce large amounts of heterologous proteins in a soluble and native form. For many applications a high purity of the recombinant protein is demanded, which implies a purification strategy where the product efficiently can be isolated from the complex milieu of host cell contaminants. In this thesis, different strategies based on both rational and combinatorial genetic engineering principles have been investigated, aiming at improving and facilitating recombinant E. coli protein production and purification. One objective was to improve the PEG/salt aqueous two-phase system (ATPS) purification process of the lipase cutinase, by increasing the selectivity of the protein for the system top-phase. Peptide tags, with varying properties, were designed and genetically fused to the C-terminal end of ZZ-cutinase. Greatly increased partitioning values were observed for purified protein variants fused to tryptophan containing peptide tags, particularly a (WP)4 peptide. The partitioning properties of the ZZ-cutinase-(WP)4 protein were also retained when added to the ATPS directly from an E. coli total cell disintegrate, emphasizing the applicability of this genetic engineering strategy for primary protein purification in ATPSs. Further on, a combinatorial library approach using phage display technology was investigated as a tool for identification of peptide tags capable of improving partitioning properties of ZZ-cutinase in an ATPS. Repeated ATPS-based partitioning-selection cycles of a large phagemid (pVIII) peptide library, resulted in isolation of phage particles preferentially decorated with peptides rich in tyrosine and proline residues. Both a peptide corresponding to a phage library derived peptide sequence as well as peptides designed based on information of amino acid appearance frequencies in later selection rounds, were shown to improve partitioning several-fold when genetically fused to the C-terminal end of ZZ-cutinase. From the two- to four–fold increased production yields observed for these fusion proteins compared to ZZ-cutinase-(WP)4, it was concluded that the selection system used allowed for selection of desired peptide properties related to both partitioning and E. coli protein production parameters. Bacterial protein production is affected by several different mRNA and protein sequence-related features. Attempts to address single parameters in this respect are difficult due to the inter-dependence of many features, for example between codon optimization and mRNA secondary structure effects. Two combinatorial expression vector libraries (ExLib1 and ExLib2) were constructed using a randomization strategy that potentially could lead to variations in many of these sequence-related features and which would allow a pragmatic search of vector variants showing positive net effects on the level of soluble protein production. ExLib1 was constructed to encode all possible synonymous codons of an eight amino acid N-terminal extension of protein Z, fused to the N-terminal of an enhanced green fluorescent reporter protein (EGFP). In ExLib2, the same eight positions were randomized using an (NNG/T) degeneracy code, which could lead to various effects on both the nucleotide and protein level, through the introduction of nucleotide sequences functional as e.g. alternative ribosome binding or translation initiation sites or as translated codons for an Nterminal extension of the target protein by a peptide sequence. Flow cytometric analyses and sorting of library cell cultures resulted in isolation of clones displaying several-fold increases in whole cell fluorescence compared to a reference clone. SDS-PAGE and western blot analyses verified that this was a result of increases (up to 24-fold) in soluble intracellular ZEGFP product protein content. Both position specific codon bias effects and the appearance of new ribosomal binding sites in the library sequences were concluded to have influenced the protein production. To explore the possibility of applying the same combinatorial library strategy for improving soluble intracellular production of heterologous proteins proven difficult to express in E. coli, three proteins with either bacterial (a transcriptional regulator (DntR)) or human (progesterone receptor ligand binding domain (PRLBD) and 11-β Hydroxysteroid dehydrogenase type I (11-β)) origin, were cloned into the ExLib2 library. Flow cytometric sorting of libraries resulted in isolation of DntR library clones showing increased soluble protein production levels and PR-LBD library clones with up to ten-fold increases in whole cell fluorescence, although the product under these conditions co-separated with the insoluble cell material.
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- Berglund, Anders, et al.
(author)
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On insights into teaching and learning technological systems
- 2016
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In: Different horizons: setting directions for phenomenographic research, EARLI Special Interest Group 9, Phenomenography and Variation Theory, Gothenburg 24-26 Aug..
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Conference paper (other academic/artistic)abstract
- The aim of the ETSC project is to explore how pupils, aged 11 - 13, learn about technological systems, with a further goal of offering insights useful for designing good teaching. This is a complicated tak as there is only is little previous research, no jointly agreed definition of the object of the pupils’ learning or how it should be taught exists, and the research on learning situations where learners work in teams with a high degree of agency is methodologically not well developed. Inspired by phenomenography and variation theory, we have collected data from lessons in technological systems. To analyse this learning we have developed a framework, where the four positions on which it is based together offer insights in the pupils’ learning, valuable for teaching. •A core position is technology itself, as it is the object of the pupils’ learning. •A classic phenomenographic analysis reveals how the pupils experience technological systems. •The teaching emphasis three dimensions of variation in understanding technological systems (Svensson, 2011). We investigate to what extent these dimensions are mirrored in the pupils’ learning. •The phenomenographic distinction on deep and surface learning offers a way to express how the pupils approach their learning. In our presentation, we will exemplify how these positions, taken together, can offer insights in the pupils’ perspective on technological systems.
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- Berglund, Sten, 1947-, et al.
(author)
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The breakdown of communism in Eastern Europe
- 1994. - 2
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In: The new democracies in Eastern Europe. - Aldershot : Edward Elgar. - 1852789328 ; , s. 1-13
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Book chapter (other academic/artistic)
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| 8. |
- Berglund, Sten, 1947-, et al.
(author)
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The failure of popular democracy
- 1994. - 2
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In: The new democracies in Eastern Europe. - Aldershot : Edward Elgar. - 1852789328 ; , s. 14-35
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Book chapter (other academic/artistic)
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| 9. |
- Berglund, Åke, et al.
(author)
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Från affärskompetens till affärsutveckling i småföretag : en studie kring processerna bakom kompetensutveckling och affärsutveckling bland småföretag i olika affärsmiljöer
- 1999
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Doctoral thesis (other academic/artistic)abstract
- The main question for this study is: How do small firms increase their level of competence? We have chosen to focus on how the conditions can be created for increasing competence, and what the connection is between business strategy and competence development. The aim of the study is two-fold. The first aim is to develop a model for the purpose of describing and understanding competence development in small firms. The study model will assist us in understanding the connection between competence development and development of the company's business strategy. The second aim is to analyze the manner in which competence is developed in small firms and how competence development is related to change in business strategy.Methodologically we start with a qualitative onset and we use a systems approach to develop a model describing the relations between the business environment, internal processes, sources of competence, business competence and change in business strategy. The empirical data used in the study consists of a relatively large amount of documentation of 32 small companies with 10 to 49 employees.An experience from the study is that small firms that have developed a greater amount of sources of competence also have changed their business concept to a larger extent. The "family businesses" usually develop fewer sources of competence and more seldom change their business strategies than other companies. One of our experiences from the study is that the company's management can influence the development of competence within the organization in many ways. By creating an organizational structure that provides the opportunity for the members within the organization to learn, either individually or in a group. By shaping the production process so that it encourages cooperation and exchange of information, sources of competence are created in the day to day operations. By expanding flexibility, that is to have a feel for the business environment, which makes it possible for the organization to acquire new competence. The management process can be adapted by increasing democracy and decentralizing, as well as by the development of competence-supporting routines.
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- Berglund, Åke, 1961-
(author)
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Optimisation of Chemotherapy Treatment in Advanced Colorectal Cancer
- 2002
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Doctoral thesis (other academic/artistic)abstract
- Colorectal cancer is one of the most common malignant diseases in Sweden – more than 5000 new cases are diagnosed each year. The overall five-year survival is about 60% and in cases of recurrence the prognosis is poor.In a phase III study in advanced colorectal cancer the response rate was doubled when 5-FU was given as a bolus injection versus as a short infusion. The toxicity was similar and time to progression was longer in the injection group. However, overall survival was not significantly different. Dose-effect relationships of 5-FU were studied in another phase III study recruiting 312 patients. A decrease from 500 mg/m2 to 400 mg/m2 worsened the treatment results. A low incidence of severe toxicity was seen in both groups. An increase to 600 mg/m2 worsened the toxicity without any improvement of the results.A cytotoxic drug sensitivity test in different tumour types, mainly gastrointestinal cancer, poorly predicted treatment outcome in a phase II study.The conventional Nordic Flv regimen was split in a phase I/II trial. An escalation of dose was possible and the response rate was 20%.Thymidylate synthase (TS) and the gene expression of p53 were investigated by immunohistochemical technique in the primary tumours of 132 patients. None of the markers predicted the later palliative chemotherapy result. However, TS significantly predicted time to recurrence.Serum markers were analysed before and during FLv treatment to early predict outcomes among 87 patients. TPS is promising, both as a predictive marker before start of treatment and after a short period of treatment. In the same setting, CEA had lower predictive value. S-VEGF and S-bFGF did not yield any prognostic information of later outcome. In all studies B-haemoglobin values, performance status and subjective response were strong markers, both for prediction of objective response and for survival.
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