| 1. |
|
|
| 2. |
- Bandmann, Nina, 1971-
(författare)
-
Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
|
|
| 3. |
- Berglund, Johan C, 1978, et al.
(författare)
-
Measuring strategies for smooth tool steel surfaces
- 2008
-
Ingår i: Proceedings. - Aachen : Shaker Verlag. - 1610-4773. - 9783832269128 - 3832269126 ; , s. 110-119
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Comparisons between different measuring strategies were made on three types of smooth tool steel surfaces. Three replica materials were tested to study possibilities within replication techniques. An optical interferometer as well as a mechanical stylus was used to evaluate the surfaces. The results showed that the tested replica materials generated good representations of both the form and the surface roughness (Sq > 300 nm). The evaluated surfaces were quite homogeneous, thus, few measurements are needed to get representative results. However, it was found that caution must be taken regarding manually polished surfaces which can be less homogenous and therefore require more measurements to get representative results.
|
|
| 4. |
- Berglund, Kerstin, 1961-
(författare)
-
Straffrätt och kön
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis starts in feminist theory and the debate on sex/gender and knowledge. There are two lines of inquiry, one in criminal law and one in feminist theory that interconnect throughout the book. Their common grounds are the depiction of the individual. In criminal law “man” is found first of all on the theoretical level as part of legal theory. Criminal law is based on the traditional liberal ideal of the autonomous individual. When criminal law is applied legal reasoning also demands an idea of what a human being is. Legal reasoning is therefore always dependent on descriptions of both “man” and “reality”. Feminist theory, in turn, deals primarily with sex/gender issues. In an attempt to structure this field of research and to highlight important differences in feminist theory, three positions are presented. The three positions are defined in terms of the ideological aspects of the sex/gender debate, and the epistemological theories that can be related to these different ideological positions.Different descriptions of “man” and “reality” can lead to profoundly different conditions for legal reasoning. In the book it is the contradiction between the idea of sex as a role played by a neutral individual, and the idea of gender as a fundamental aspect of human life, that is used as a starting point for the analyses of legal arguments. One question that is raised is in what way the understanding of harm to the individual changes when the conditions for describing “man” are altered. In order to answer this question, selected committee reports on rape and physical abuse of women during the period 1958 to 2001, are analyzed. All of these are to various extents related to the question of harm to the individual. When judging harm in the given examples, the gendered individual is used as an alternative way of describing “man”. The study concludes that it is important for criminal law to recognize that women are sexually abused because they are women. This is fundamental to the way in which these crimes must be interpreted. But it is also important to stress that women are sexually abused in their capacity of being women. It is argued that this constitutes the very basis for understanding harm to the individual in these cases. Victims of sexual violence are always embodied, gendered and socially situated. It is therefore important to find ways to define harm to the gendered individual. In brief, the conflicts surrounding criminal law today can be understood as the dichotomy between the liberal ideal of the autonomous individual and the feminist ideology of difference. It is therefore argued that there is a need for an ethical theory that includes the gendered individual.
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
|
|
| 9. |
- Eriksson, Ulrika, 1974-
(författare)
-
Impact of autocrine factors on physiology and productivity in Trichoplusia ni serum-free cultures
- 2005
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells.Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation.Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics.In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity.Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well.
|
|
| 10. |
- Hammarström, Martin, 1973-
(författare)
-
Protein production and purification in structural genomics
- 2006
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The number of gene products available for structural and functional study is increasing at an unprecedented rate as a result of the successful whole genome sequencing projects. Systematic structure determination of proteins on a genomic scale, called structural genomics, can significantly contribute to the field of protein science and to functional annotation of newly identified genes. This thesis covers different aspects of protein production in Eschericiha coli for structural studies in the context of structural genomics. Protocols have been downscaled and standardized to allow for a rapid assessment of the production characteristics for multiple proteins in parallel under a number of different conditions. Foremost, the ability of different proteins and peptide tags to affect the solubility of the recombinant protein when produced as fusion proteins has been systematically studied. Large differences in the success-rate for production of soluble protein in E. coli were found depending on the fusion partner used, with a more than two-fold increase in the number of proteins produced as soluble when comparing the best and the poorest fusion tags. For different constructs with a histidine tag, commonly used to facilitate protein purification, large differences in yield depending on the design of the expression vector were found. When comparing different fusion proteins produced from identical expression vectors, fusions to the GB1 domain were found to result in the highest yield of purified target protein, on average 25 % higher than any of the other fusions. The suitability for further structural studies was tested at an intermediate scale for proteins that were identified as soluble in the expression screening. For this purpose, protocols for rapid purification and biophysical characterization using nuclear magnetic resonance and circular dichroism spectroscopy were developed and tested on 19 proteins, of which four were structured.
|
|
