<p>The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein.</p><p>The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Z_basic1, Z_acid2and Z_trp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (<=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases.</p><p>Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems.</p><p>The capture ofß-galactosidase from<i>E. coli</i>cell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load.</p><p><b>Keywords:</b><i>E. coli</i>, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags</p>

Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity. The wild-type North American firefly(Photinus pyralis)luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C. The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible. Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c7dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS). Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions. A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU. Key words:bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c7dATP, dATPαS. / <p>QCR 20161027</p>

Nucleoside 5-triphosphates are the building blocks to synthesis of nucleic acids. Nucleic acids (RNA and DNA) participate in many important biological functions in living systems, including genetic information storage, gene expression, and catalysis. Nucleoside 5- triphosphates have many important therapeutic and diagnostic applications. To understand how these triphosphates are utilized in living systems, numerous synthetic mimics have been prepared and used as active metabolites of certain drugs and molecular probes. Over the years, nucleic acids have been modified at the nucleobase, sugar moiety and phosphate backbone with the aim of understanding their structures and functions. We have site-specifically replaced selected oxygen atoms of nucleosides and nucleotides with selenium atom in order to enzymatically synthesize selenium-derivatized DNAs for obtaining insights into the DNA flexibility, duplex recognition and stability. Although triphosphates have important biological and medicinal significance, they are however, very difficult to synthesize and isolate in high purity and yield. There are many approaches to the synthesis of nucleoside 5-triphosphates, but there is no general strategy that allows simple and direct synthesis of nucleoside triphosphates. To face the challenges, we have developed a new approach in the absence of protecting groups to quickly and efficiently synthesized native deoxynucleoside 5-triphosphates and deoxynucleoside 5-(α- P-seleno)- P-seleno)triphosphates. Syntheses of the triphosphates containing selenium-derivatized nucleobases were also successfully accomplished. After replacing the oxygen atoms at the 4-position of thymidine and uridine, and the 6-position of guanosine, we observed most strikingly, a large bathrochromic shift of over 100 nm, relative to their native counterparts of UV absorbance of 260 nm. Consequently, the synthesized selenium base modified triphosphates are yellow. We also synthesized 2-selenothymidine and 5-methylseleno thymidine 5-triphosphates. We conducted stability study on the colored 4-selenothymidine and used the 5- triphosphate analog (4-SeTTP) as substrate for polymerase recognition. The Klenow polymerase incorporated the 4-SeTTP with efficiency equal to that of the native counterpart. Finally, 4-SeTTP was used to demonstrate UVdamage resistance of selenium-derivatized DNAs and plasmid.

<p>Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity.</p><p>The wild-type North American firefly<i>(Photinus pyralis)</i>luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C.</p><p>The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible.</p><p>Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c⁷dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS).</p><p>Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions.</p><p>A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU.</p><p><b>Key words:</b>bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c⁷dATP, dATPαS.</p>

The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein. The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Zbasic1, Zacid2and Ztrp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (&lt;=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases. Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems. The capture ofß-galactosidase fromE. colicell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load. <b>Keywords:</b>E. coli, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags / NR 20140805