| 1. |
- 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.
|
|
| 2. |
- 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.
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
- Höst, Gunnar, 1976-
(författare)
-
Engineering carbonic anhydrase for highly selective ester hydrolysis
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- I denna avhandling presenteras arbete utfört med enzymet humant karboanhydras II (HCAII). Enzymer är en typ av proteiner som accelererar (katalyserar) kemiska reaktioner, vilket är nödvändigt för allt levande. Den naturliga funktionen för HCAII är att katalysera omvandlingen av gasen koldioxid till vätekarbonat, som är löslig i vätska. Detta är viktigt bl.a. för att koldioxid som bildas i kroppen, och fraktas i blodet i form av vätekarbonat, skall hinna över till utandningsluften under den korta tid blodet är i lungorna.Proteiner består av aminosyror som länkats samman i en lång kedja, där varje aminosyra är en av de 20 naturliga aminosyratyperna. Ett proteins struktur och egenskaper bestäms av aminosyrasekvensen, som i sin tur bestäms av genen för just det proteinet. Med genteknik kan ett proteins gen ändras (muteras), så att aminosyrasekvensen ändras, och det har här utnyttjats för att förändra HCAIIs katalytiska egenskaper. Förutom dess naturliga funktion kan HCAII även klyva (hydrolysera) vissa estrar. Mutationer gjordes så att en ’ficka’ i HCAIIs struktur, där molekylerna (substraten) som skall klyvas binder, fick en större volym. På så sätt skapades varianter med en kraftigt ökad kapacitet för att hydrolysera långa estersubstrat jämfört med icke-muterat HCAII. Som en utveckling av detta projekt skapades en mutant av HCAII, som kan hydrolysera ett än mer skrymmande substrat.I ett annat projekt har en ny katalytisk aktivitet skapats i HCAII, som inte utnyttjar enzymets naturliga katalytiska förmåga. Ett nytt estersubstrat konstruerades, med en del som binder kraftigt till HCAII, så att en stark substratbindning erhölls. Sedan muterades vissa aminosyror till en reaktiv aminosyra som heter histidin. Valet av positioner för mutation baserades på en datormodell av enzymet med bundet substrat. Eftersom histidin kan delta i hydrolysreaktioner, får det muterade enzymet möjlighet att klyva substratet. Flera olika mutanter testades, och den effektivaste innehöll ett nära kopplat par av histidiner. Denna mutant undersöktes mer noggrannt, vilket gav viss information om den katalytiska mekanismen.Det långsiktiga målet med detta arbete är att konstruera muterade enzymer som kan klyva giftiga ämnen, eller användas vid framställning av kemikalier. Det finns behov av nya enzymer för olika typer av substrat, och att med rationella metoder skapa nya katalytiska aktiviteter i proteiner är ett svårt vetenskapligt problem som ännu är i ett tidigt utvecklingsskede.
|
|
| 6. |
- Ljungdahl, Jonas, 1979-
(författare)
-
Structure and properties of Vasa oak
- 2006
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Vasa ship is not adequately supported. Measurements of the hull show that the ship deforms and rotate towards the port side. In addition, damages on the hull at support areas have been observed. The damages are due to high compressive loads. At damaged zones the support has been removed and the loads are thus transferred to adjacent support stanchions. In order to design an improved support, knowledge of the mechanical behaviour of the material is needed. In particular, radial modulus, strength and deformation mechanisms are of interest. In the present study, the mechanical behaviour of recent oak and oak from Vasa is studied. Furthermore, effects of PEG content, degradation and moisture on the properties of Vasa oak are investigated. Oak is characterized by a very abrupt change from earlywood to latewood, where the latewood is much denser than earlywood. Also present in oak are large rays in the radial direction of the wood. Small specimens were tested in compression using Digital Speckle Photography (DSP) in order to obtain strain fields of the whole specimen surface. This technique also provided data on failure mechanisms. Dynamic mechanical thermal analysis (DMTA) was performed to establish differences in moisture softening. In radial compression, modulus and strength of Vasa oak are reduced by 50% compared with recent oak. A significant change of failure mechanism is observed for Vasa oak. In recent oak, failure in radial compression is by continuous folds of rays in the earlywood followed by continued plastic collapse of the earlywood layer. In Vasa oak rays show a more brittle fracture in each earlywood region. DMTA results indicate no effect on moisture softening of Vasa oak from presence of PEG although more work is needed to confirm this. Moisture adsorption for PEG-extracted Vasa oak is not significantly higher than for recent oak below 60% RH, suggesting that the extent of degradation of Vasa oak is limited. Vasa oak containing PEG is much more hygroscopic than PEG-extracted Vasa oak already at 50%. This difference is increasing with increasing relative humidity.
|
|
| 7. |
- Marx, Lisa, 1990-
(författare)
-
Chemo-enzymatic cascades for the synthesis of chiral high-value chemicals
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chiral amines are frequent in today´s top selling pharmaceuticals. Classical organic synthesis of pharmaceuticals is often work intensive involving many synthesis steps, the use of protection group chemistry, heavy metal catalysts and chiral crystallization techniques. In recent years biocatalysts have proven their outstanding ability to synthesize chiral compounds. In this work the possibility of employing biocatalysts as alternative catalysts for API (active pharmaceutical ingredient) synthesis was explored. Three compounds currently on the market were selected as viable case studies: Cinacalcet (a hyperparathyroidism drug), Vyvanse (an ADHD-drug) and Sertraline (an antidepressant). Two enzyme classes were investigated to directly provide the chiral amines - transaminases and imine reductases. Ketoreductases were also investigated to provide the chiral amine via the chiral alcohol. Laccases and hydrolases were employed to complete the synthesis pathways to the final API. In the case of Vyvanse a true one-pot, two-step enzymatic cascade was achieved by a transaminase and hydrolase. For Cinacalcet a chemo-enzymatic cascade could be demonstrated. Both transaminase and ketoreductase gave excellent enantioselectivities and high yield for the key intermediates, which could then be chemically converted into the final API with good yield. For Sertraline the best yield of one diastereomer precursor could be achieved by a ketoreductase, followed by further enzymatic and chemical steps to the final API. Transaminases and imine reductases both have potential in synthesizing the key amine precursors or the APIs themselves. But to date selectivity and yield are insufficient for industrial application in a lot of cases. This work demonstrates the potential of enzymes to serve as viable alternatives to organo-metallic synthesis. Furthermore enzymes have the potential to simplify work-up because of their excellent enantioselectivity. Finally, a scale-up of a one-step transamination to the key chiral precursor of Cinacalcet demonstrated the enzyme´s applicability in larger volume and at higher substrate concentration.
|
|
| 8. |
|
|
| 9. |
|
|
| 10. |
|
|
