The innovation of DNA analysis techniques has enabled arevolution in the field of molecular biology. In the 70’s,first technologies for sequence determination of DNA wereinvented and these techniques enormously increased thepossibilities of genetic research. A large proportion ofmethods for DNA sequencing is based on enzymatic DNA synthesiswith chain termination followed by electrophoretic separationand detection. However, alternative approaches have beendeveloped and one example of this is the pyrosequencingtechnology, which a four-enzyme DNA sequencing method based onreal-time monitoring of DNA synthesis. Currently, the method is limited to analysis of short DNAsequences and therefore it has primarily been used for mutationdetection and single-nucleotide polymorphism analysis. In orderto expand the use of the pyrosequencing technology, the readlength obtained in the methods needs to be improved. However,it was previously shown that the data quality in pyrosequencingtechnology could be significantly increased by addition ofEscherichia coli single-stranded DNAbinding protein, SSB, tothe sequencing reaction. Since little was known about themechanism of this enhancement, we performed a systematic effortto analyse the effect of SSB on 103 clones randomly selectedfrom a cDNA library. We investigated the effect of SSB on theobtained read length in pyrosequencing and identified thecauses of low quality sequences. Moreover, the effciency ofprimer annealing and SSB binding for individual cDNA clones wasinvestigated by use of real-time biosensor analysis. Resultsfrom these experiments show that templates with highperformance in pyrosequencing without SSB possess effcientprimer annealing and low SSB affnity. To minimise the cost of the pyrosequencing system, effcientand scaleable procedures for production and isolation of theprotein components are required. Therefore, protocol foreffcient expression in E.coliand rapid isolation of native SSB was developed.Moreover, by use of a gene fusion strategy, Klenow polymerasewas produced in fusion with the Zbasic domain at high levels inE. coli. This highly charged protein handle enables selectiveand effcient ion exchange purification at physiological pH.Furthermore, active Apyrase was expressed in Methyltropic yeastPichia pastoris and purified by two chromatographic steps. Since pyrosequencing analysis mainly is performed in a96-sample plate format, an increase in sample capacity would bevery beneficial. One approach to achieve this would be to usemicromachined filter chamber arrays where nano-liter samplescan be monitored in real-time. However, to enable accuratepyrosequencing analysis of parallel samples, the produced lightshould preferable be docked to the correct DNA template.Therefore, two different gene fusion strategies were utilisedbased on directed immobilisation of the light-harvesting enzymeLuciferase on the DNA molecules. The thermostable variant ofthe enzyme was genetically fused to a DNA binding protein(either SSB or Klenow) and the Zbasic purification handle, which could beselectively removed by protease cleavage. A protocol wasdeveloped for effcient expression in E.coliand purification by Ion Exchange Chromatography.The proteins were analysed by complete extension of DNAtemplates immobilised on magnetic beadspyrosequencing monitoredby pyrosequencing chemistry. Results from these experimentsshow that the proteins bound selectively to the immobilised DNAand that their enzymatic domains were active. In summary, the work presented in this thesis pinpointsfeatures in the pyrosequencing technology that needs to befurther developed. Moreover, various protein-based strategiesare presented in order to overcome these limitations. <b>Keywords:</b>pyrosequencing, SSB, Zbasic, Klenow, Apyrase, expression, purification,Biacore, DNA template length, Luciferase, affnity, gene fusion,immobilisation.

<p>The innovation of DNA analysis techniques has enabled arevolution in the field of molecular biology. In the 70’s,first technologies for sequence determination of DNA wereinvented and these techniques enormously increased thepossibilities of genetic research. A large proportion ofmethods for DNA sequencing is based on enzymatic DNA synthesiswith chain termination followed by electrophoretic separationand detection. However, alternative approaches have beendeveloped and one example of this is the pyrosequencingtechnology, which a four-enzyme DNA sequencing method based onreal-time monitoring of DNA synthesis.</p><p>Currently, the method is limited to analysis of short DNAsequences and therefore it has primarily been used for mutationdetection and single-nucleotide polymorphism analysis. In orderto expand the use of the pyrosequencing technology, the readlength obtained in the methods needs to be improved. However,it was previously shown that the data quality in pyrosequencingtechnology could be significantly increased by addition ofEscherichia coli single-stranded DNAbinding protein, SSB, tothe sequencing reaction. Since little was known about themechanism of this enhancement, we performed a systematic effortto analyse the effect of SSB on 103 clones randomly selectedfrom a cDNA library. We investigated the effect of SSB on theobtained read length in pyrosequencing and identified thecauses of low quality sequences. Moreover, the effciency ofprimer annealing and SSB binding for individual cDNA clones wasinvestigated by use of real-time biosensor analysis. Resultsfrom these experiments show that templates with highperformance in pyrosequencing without SSB possess effcientprimer annealing and low SSB affnity.</p><p>To minimise the cost of the pyrosequencing system, effcientand scaleable procedures for production and isolation of theprotein components are required. Therefore, protocol foreffcient expression in E.<i>coli</i>and rapid isolation of native SSB was developed.Moreover, by use of a gene fusion strategy, Klenow polymerasewas produced in fusion with the Zbasic domain at high levels inE. coli. This highly charged protein handle enables selectiveand effcient ion exchange purification at physiological pH.Furthermore, active Apyrase was expressed in Methyltropic yeastPichia pastoris and purified by two chromatographic steps.</p><p>Since pyrosequencing analysis mainly is performed in a96-sample plate format, an increase in sample capacity would bevery beneficial. One approach to achieve this would be to usemicromachined filter chamber arrays where nano-liter samplescan be monitored in real-time. However, to enable accuratepyrosequencing analysis of parallel samples, the produced lightshould preferable be docked to the correct DNA template.Therefore, two different gene fusion strategies were utilisedbased on directed immobilisation of the light-harvesting enzymeLuciferase on the DNA molecules. The thermostable variant ofthe enzyme was genetically fused to a DNA binding protein(either SSB or Klenow) and the Z_basic purification handle, which could beselectively removed by protease cleavage. A protocol wasdeveloped for effcient expression in E.<i>coli</i>and purification by Ion Exchange Chromatography.The proteins were analysed by complete extension of DNAtemplates immobilised on magnetic beadspyrosequencing monitoredby pyrosequencing chemistry. Results from these experimentsshow that the proteins bound selectively to the immobilised DNAand that their enzymatic domains were active.</p><p>In summary, the work presented in this thesis pinpointsfeatures in the pyrosequencing technology that needs to befurther developed. Moreover, various protein-based strategiesare presented in order to overcome these limitations.</p><p><b>Keywords:</b>pyrosequencing, SSB, Z_basic, Klenow, Apyrase, expression, purification,Biacore, DNA template length, Luciferase, affnity, gene fusion,immobilisation.</p>

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HTML clipboard The great advances in DNA technology, e.g. sequencing and recombinant DNA techniques, have given us the genetic information and the tools needed to effectively produce recombinant proteins. Recombinant proteins are valuable means in biotechnological applications and are also emerging as alternatives in therapeutic applications. Traditionally, monoclonal antibodies have been the natural choice for biotechnological and therapeutic applications due to their ability to bind a huge range of different molecules and their natural good affinity. However, the large size of antibodies (150 kDa) limits tissue penetration and the recombinant expression is complicated. Therefore, alternative binders with smaller sizes have been derived from antibodies and alternative scaffolds. In this thesis, two structurally similar domains, Zbasic and ABDz1, have been used as purification tags in different contexts. They are both three-helical bundles and derived from bacterial surface domains, but share no sequence homology. Furthermore, by redesign of the scaffold used for ABDz1, a molecule intended for drug targeting with extended in-vivo half-life has been engineered. In Papers I and II, the poly-cationic tag Zbasic is explored and evaluated. Paper I describes the successful investigation of Zbasic as a purification handle under denaturating conditions. Moreover, Zbasic is evaluated as an interaction domain in matrixassisted refolding. Two different proteins were successfully refolded using the same setup without individual optimization. In Paper II, Zbasic is further explored as a purification handle under non-native conditions in a multi-parallel setup. In total, 22 proteins with varying characteristics are successfully purified using a multi-parallel protein purification protocol and a robotic system. Without modifications, the system can purify up to 60 proteins without manual handling. Paper I and II clearly demonstrate that Zbasic can be used as an interaction domain in matrix-assisted refolding and that it offers a good alternative to the commonly used His6-tag under denaturating conditions. In paper III, the small bifunctional ABDz1 is selected from a phage display library. Endowed with two different binding interfaces, ABDz1 is capable of binding both the HSA-sepharose and the protein A-derived MabSelect SuRe-matrix. The bifunctionality of the domain is exploited in an orthogonal affinity setup. Three target proteins are successfully purified using the HSA-matrix and the MabSelect SuRe-matrix. Furthermore, the purity of the target proteins is effectively improved by combining the two chromatographic steps. Thus, paper III shows that the small ABDz1 can be used as an effective purification handle and dual affinity tag without target specific optimization. Paper IV describes the selection and affinity maturation of small bispecific drug-targeting molecules. First generation binders against tumor necrosis factor-α are selected using phage display. Thereafter on-cell surface display and flow cytometry is used to select second-generation binders. The binding to tumor necrosis factor-α is improved up to 30 times as compared to the best first generation binder, and a 6-fold improvement of the binding strength was possible with retained HSA affinity. Paper III and IV clearly demonstrate that dual interaction surfaces can successfully be grafted on a small proteinaceous domain, and that the strategy in paper IV can be used for dual selection of bifunctional binders. / <p>QC20100610</p>

The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli. A positively charged purification tag, Zbasic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Zbasic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified Escherichia coli homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Zbasic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Zbasic, was effected by adsorption to a second cation-exchanger. Using a similar strategy, a purification tag with a negatively charged surface, denoted Zacid, was constructed and thoroughly characterised. Contrary to Zbasic, the negatively charged Zacid was highly unstructured in a low conductivity environment. Despite this, all Zacid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed in vivo by the use of a flow cytometer. The positively charged domain, Zbasic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Zbasic as a reversible linker to the cation-exchanger resin. / QC 20101020

<p>The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli.</p><p>A positively charged purification tag, Z_basic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Z_basic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified <i>Escherichia coli</i> homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Z_basic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Z_basic, was effected by adsorption to a second cation-exchanger. </p><p>Using a similar strategy, a purification tag with a negatively charged surface, denoted Z_acid, was constructed and thoroughly characterised. Contrary to Z_basic, the negatively charged Z_acid was highly unstructured in a low conductivity environment. Despite this, all Z_acid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography</p><p>Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed <i>in vivo</i> by the use of a flow cytometer. </p><p>The positively charged domain, Z_basic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Z_basic as a reversible linker to the cation-exchanger resin.</p>