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1	Uttryck av ett nytt rekombinant protein Cp149 (HBV-kapsidprotein) modifierat med TfR apical domän / Expression of a new recombinant protein Cp149 (HBV capsid protein) modified with TfR apical domain Noorzai, Hamida January 2022 (has links) Hepatit-B är en leversjukdom som orsakas av hepatit-B-virus (HBV) vilket är en kapslad DNA-virus. Kapsidprotein (Cp) har stor betydelse i virusets livscykel exempelvis DNA-replikation, interaktion med värdceller och andra virala glykoproteiner. HBV, som många andra virus, tar sig in i cellen genom att binda till cellreceptorer. Transferrinreceptor är en välkänd receptor som mögliggör virus inträde i cellen genom att binda till virusproteiner, intraktionen sker i apikala domänen i TfR. Båda Cp149, kapsidsammansättnings domänen i Cp, och apikala domänen i TfR är betydelsefulla ändamål för utveckling av antivirala läkemedel. Syftet med arbetet var att klona och uttrycka olika varianter av ett nytt modifierat Cp149, där Cp149 har modifierats med AP01 (lösliga formen av apikala dömanen), och analysera intraktioner mellan proteinerna och viralt glykoprotein, MGP1. Modifierade proteingener klonades i plasmid (pET-11a) med hjälp av rekombinant DNA-teknik och användning av restriktionsenzymer NdeI och BamHI. Agarosgelelektrofores och DNA-sekvensering användes för att kontrollera förekomst av eftersökta DNA-sekvenser. Nya plasmider fördes över till bakterieceller, Escherichia coli, och proteinutrycket inducerades i bakteriecellerna genom kemiskbehandling. Framrenade proteiner från respektive provlösning analyserades med Sodium dodecyl sulphate polyakrylamid gel electrophoresis (SDS-PAGE) och proteinernas funktion undersöktes med flödescytometri genom att besämma bindningsförmågan till MGP1, som uttrycktes på jästceller, i närvaro av TfR. Rekombinant plasmid innehållande proteingen kodande Cp149 för varianter A-D samt F lyckades att framställas. Resultatet från SDS-PAGE påvisade inga tydlyga protein-band och flödescytometri resultatet var svårt att bedömma, troligen då ytterliggare proteinupprening behövdes för att isolera kapsidproteinerna. Syftet med arbetet har erhållits delvis och fortsatt undersökningar på nya proteiner förslås. / Hepatitis B is one av the major worldwide health problems that is caused by enveloped DNA virus, Hepatitis B virus (HBV). HBV’s capsid protein (Cp) has an important role in the virus life cycle, för example DNA replication, intraction with host cells, and other viral glycoproteins. HBV, like many other viruses, enters the cells by binding to cell receptors. Transferrin receptor (TfR) is a well-known receptor that enables virus entry into the cell by binding to viral proteins. The interaction takes place in the apical domain of TfR. Both Cp149, the capsid’s composition domain of Cp, and the soluble form of the apical domain, AP01, from TfR are important builing parts to be explored as starting building blocks for the development of antiviral therapeutics. Modification of Cp149 with AP01 is an interesting combination to produce new protein-based drugs to prevent viral infections. The aim of this project was to clone and express six different variants of a AP01 modified Cp149 protein as a starting points to analyze interactions between the proteins and Machupo virus glykoprotein 1 (MGP1). All target DNA templates and plasmids (pET-11a) were digested with restriction enzymes, NdeI and BamHI, and ligated by T4 DNA ligase. Agarose gel electrophoresis and DNA sequencing were used as validation methods to confirm the presence of desired and corrected DNA sequences, respectively, during gene cloning. DNA transformation and induction of Escherichia coli cells was used to express the desired proteins. The purified proteins were validated for their binding ability to MGP1, expressed on yeast cells by flow cytometry in a competition assay with TfR. Recombinant plasmid including the expected DNA sequence encoding Cp149 for variants A-D and F was successfully produced. There was no clear detection of protein bands on Sodium dodecyl sulphate polyakrylamid gel electrophoresis (SDS-PAGE) gel and flow cytometry results were difficult to interpret due to insufficient protein purification during ammonium sulphate percipitation. The purpose of the project has been obtained partially and more studies have to be carried out to produce pure proteins that can be used for further analysis. Gene cloning Recombinant DNA Transformation Protein expression SDS-PAGE Flow Cytometry Genkloning rekombinant DNA Transformering Proteinuttryck SDS-PAGE Flödescytometri Biomedical Laboratory Science/Technology
2	Scale-down modelling of the upstream process for production of Affibody® Molecules Masreliez, Philip January 2022 (has links) I detta projekt har uttrycket av Affibodymolekyler i bioreaktorer av olika volymetriska skalor jämförts för att fastställa om ett tillförlitligt samband mellan de olika bioreaktorernas prestanda kan etableras för att möjliggöra utvecklingen av en nedskalad produktionsmodell för Affibodymolekyler. Baslinjen för jämförelsen i denna studie har varit en enliters- stirred tank reactor (STR) som de andra (mindre) bioreaktorernas prestanda jämfördes med. Jämförelsen av prestanda gjordes genom odling och uttryck av sex olika Affibodymolekyler i replikat i varje bioreaktorstorlek. Prestandan i detta fall hänvisar till produktionen av Affibodymolekyler (mg/L Cellodling) under odlingen, som fastställdes genom ett protokoll för proteinrening av lösligt intracellulärt protein genom affinitetskromatografi och kvantifiering genom absorbans vid 280 nm. De sex olika Affibodymolekyler som har studerats i detta projekt hade tidigare visat sig ha olika uttrycksnivåer, och har här jämförts med varandra i de olika bioreaktorskalorna. De två olika bioreaktorstorlekarna som bedömdes var en 300 mL skakkolv med 50 mL arbetsvolym och en mikrotiterplatta (MTP) med 3 mL arbetsvolym. Dessutom innebar studien en bedömning av två system för en långsam frisättning av kolkälla i odlingarna, ett i varje nedskalad bioreaktorstorlek. Detta utfördes för att delvis efterlikna den kontrollerade fed-batch-kulturen i STR, där koltillförsel kontrollerades med hjälp av en feedprofil. Resultaten visade att proteinuttrycket av metoderna med en långsam frisättning av kolkällor överensstämde närmast med proteinuttrycket i STR. Anpassningen av dessa resultat mot proteinuttrycket hos STR gav i en linjär regression ett R2 på 99,69 % i 3 ml MTP och 97,46 % i 50 ml skakkolven. Slutsatserna som drogs var att SMFP08003 FeedPlate var den bästa kandidaten för en nedskalad modellering av uppströmsprocessen för produktion av Affibodymolekyler. Samt att faktorn att använda en fed-batch-process istället för en batch-process har en större inverkan på proteinuttrycket än skalan av processerna. / In this project the expression of Affibody® molecules in bioreactors of different volumetric scales has been compared, to determine if a reliable relation between the performance of the different bioreactors can be established to allow for the development of a scale-down Affibody® molecule production protocol. The baseline of comparison in this study has been a one litre Stirred Tank Reactor (STR) to which the other (smaller) bioreactors' performance were compared. The performance comparison was achieved by the cultivation and subsequent expression of six different Affibody® molecules in replicates in each bioreactor size. Performance in this case refers to Affibody® molecule production (mg/L culture) during the cultivation, which is assessed by a protocol of protein purification of soluble intracellular protein by affinity chromatography and quantification by 280 nm absorbance. The six different Affibody® molecules studied in this project had previously been found to have different expression levels, and were in this project compared to each other in the different bioreactor scales. The two different bioreactor sizes which were assessed were a 300 mL shake flask with 50 mL working volume and a 3 mL working volume microtiter plate (MTP). In addition, the study involved an assessment of the use of two systems for a slow carbon source release in the cultivations, one in each scale-down bioreactor size. This was performed to partly mimic the controlled feed systems in STRs. The results showed that the protein expression of the methods with a slow carbon source release corresponded most closely with the protein expression in the STR. The fit of these results onto the protein expression of the STR yielded a R2 of 99.69% in the 3 mL MTP and 97.46% in the 50 mL shake flask. The conclusions drawn were that SMFP08003 FeedPlate was the best candidate for scale-down modelling of the upstream process for production of Affibody® Molecules and that the factor of using a fed-batch process instead of a batch process has a larger impact on the protein expression than the scale of either process. Scale-down modelling protein expression Affibody® molecule FeedPlate® EnPresso® B STR Nedskalad produktionsmodell proteinuttryck Affibodymolekyler FeedPlate® EnPresso® B STR Industrial Biotechnology Industriell bioteknik
3	Framtidens expressionssystem för svåruttryckta proteiner : Utvärdering av tolv expressionssystem / The future's expression systems for complex proteins : Evaluation of twelve expression systems Andersson, Pontus, Edenståhl, Selma, Eriksson, Elin, Hävermark, Tora, Nielsen, Jonas, Pihlblad, Alma January 2018 (has links) Today, recombinant expression of proteins is used for a variety of purposes. One of these is the production of allergens, which are vital components in allergy diagnostics. However, traditional expression systems such as Escherichia coli and Pichia pastoris might not have the capacity to express all proteins of interest. Thermo Fisher, which is a leading producer of allergy tests, has requested an evaluation of different microorganisms and their capacity for heterologous protein expression in order to expand their existing toolbox of expression systems. This summary was made through a literature study, where twelve organisms were evaluated. Six eukaryotic and six prokaryotic expression systems are compared based on their ability to properly glycosylate protein, need for specific culture conditions, safety, protease activity, duration, protein yield and protein solubility. The prokaryotic systems – Corynebacterium glutamicum , Lactococcus lactis , Pseudomonas fluorescens , Pseudoalteromonas haloplanktis , Ralstonia eutropha and Streptomyces lividans – are characterized by being easy to cultivate, operating in different temperature ranges and providing relatively high yields of recombinant protein. The eukaryotic systems – Aspergillus fungi, the green algae Chlamydomonas reinhardtii , the yeast Hansenula polymorpha , the parasite Leishmania tarentolae , the moss Physcomitrella patens and suspension-based plant cells – all have very different morphology and properties. In comparison with the prokaryotic systems, it can be concluded that they are generally better at folding and providing the correct glycosylation patterns for mammalian and plant proteins. However, they require more time and effort to establish a competent cell line. Furthermore, the resulting protein yield is usually less than for the prokaryotic systems. The conclusion can be drawn that no expression system is perfect. The solution is a toolbox, containing various expression systems and vector systems, providing the basis for successful expression of all kinds of complex proteins. Based on the evaluation of expression systems in this review, such toolbox can be obtained. Expressionssystem proteinuttryck Corynebacterium glutamicum Lactococcus lactis Pseudomonas fluorescens Pseudoalteromonas haloplanktis Ralstonia eutropha Streptomyces lividans Aspergillus Chlamydomonas reinhardtii Hansenula polymorpha Leishmania tarentolae Physcomitrella patens suspensionsbaserade växtceller Engineering and Technology Teknik och teknologier
4	Profiling the Blood Proteome in Autoimmune Disease Using Proximity Extension Assay / Profilering av blod-proteomet i autoimmuna sjukdomar genom proximity extension assay Asp, Julia January 2023 (has links) Autoimmuna sjukdomar är en samling komplexa, kroniska, inflammatoriska sjukdomstillstånd som kännetecknas av dysreglering av immunsystemet, vilket resulterar i inflammation och skada av vävnader, celler och organ. Dessa sjukdomar har en betydande inverkan på individens livskvalitet och bidrar ofta till ökad dödsrisk där komorbiditeter föreligger. Emellertid medför den varierande symptombilden för olika autoimmuna sjukdomar betydande utmaningar för att uppnå noggrann diagnos, prognos och utvärdering av behandling. Det finns därför ett påtagligt behov av att upptäcka nya biomarkörer. I denna studie utfördes en omfattande analys av 944 plasmaprover med hjälp av OlinkR Explore-plattformen, vilket genererade data för 1463 unika proteiner. Baserat på uttrycksdata identifierades proteiner förknippade med de sex utvalda autoimmuna sjukdomarna multipel skleros, myosit, reumatoid artrit, systemisk skleros, Sjögrens sjukdom och systemisk lupus erythematosus samt några av deras definierade subgrupper. Dessa potentiella biomarkörer kommer eventuellt att underlätta tidig diagnos, sjukdomsdifferentiering och prognos. Flertalet av dessa proteiner har ännu aldrig kopplats till de här specifika sjukdomarna i litteraturen, särskilt inte från plasmaprover, vilket ger spännande nya perspektiv för biomarkörsutveckling. Det är dock av största vikt att genomföra robusta valideringsstudier i oberoende kohorter. Sammanfattningsvis belyser våra resultat den potentiella brukbarheten hos dessa proteomiska plasmabiomarkörer för att förbättra tidig sjukdomsdetektering, karakterisering av subgrupper och sjukdomsdifferentiering att stimulera. Förhoppningsvis kan dessa resultat stimulera till vidare forskning inom området för biomarkörer och potentiella framsteg inom individbaserad medicin. / Autoimmune diseases are complex, chronic, inflammatory conditions characterized by dysregulation of the immune system, resulting in inflammation and damage to various tissues, cells and organs. These diseases significantly impact individuals’ quality of life and often contribute to increased mortality risk in the presence of comorbidities. However, due to the diverse array of symptoms associated with different autoimmune diseases, accurate diagnosis, prognosis, and treatment evaluation pose significant challenges. Thus, there is a pressing need for the discovery of novel biomarkers. In this study, a comprehensive analysis of 944 plasma samples using the OlinkR Explore platform was conducted, generating data on 1463 unique proteins. Based on the expression data, associated proteins were identified for six selected autoimmune diseases, namely multiple sclerosis, myositis, rheumatoid arthritis, systemic sclerosis, Sjögren’s syndrome, and systemic lupus erythematosus, as well as some of their defined subgroups. These are prospective biomarkers and have the potential to aid in early diagnosis, therapeutic intervention, subgroup identification, disease differentiation, and disease prognosis. Notably, some of these proteins have not been previously associated with the specific diseases in the existing literature, especially not in plasma samples, thereby offering intriguing new perspectives for biomarker development. However, it is of great importance to conduct robust validation studies in independent cohorts to confirm the outcomes of this study. In summary, our findings highlight the potential utility of these proteomic plasma biomarkers in improving the early detection, subgroup characterization, and disease differentiation of autoimmune diseases. The identification of these proteins will hopefully stimulate further investigation in the field of biomarker research and potential advancements in personalized medicine. Plasma proteomics proximity extension assay autoimmune disease differential expression machine learning biomarkers Plasma proteomik proximity extension assay autoimmuna sjukdomar differentiellt proteinuttryck maskininlärning biomarkörer
5	Purification, functional characterization and crystallization of the PerR peroxide sensor from Saccharopolyspora erythraea Elison Kalman, Grim January 2019 (has links) This report summarizes the work on the cloning, expression, and purification of PerR, a metal sensing regulator from Saccharopolyspora erythraea and the subsequent characterization using small angle X-ray scattering and other biochemical methods. The report aims to provide an insight into prokaryotic metal homeostasis, provide a better understanding of how PerR works and provide valuable information for the continued work on the crystallization of PerR. structural biology cell culture protein expression genetic engineering spectroscopy X-ray crystallography small angle X-ray scattering SAXS transcription factor PerR metal binding antibiotics Saccharopolyspora erythraea S. erythraea soil bacterium actinobacteria actinomycetes oxidative stress peroxide stress structure determination characterization stabilization peroxide sensor iron sensor manganese sensor metalloprotein metal ion homeostasis metal ion prokaryotic strukturbiologi cellodling proteinuttryck genteknik spektroskopi röntgenkristallografi småvinkel-röntgenspridning SAXS transkriptionsfaktor PerR metallbindande antibiotika Saccharopolyspora erythraea S. erythraea jordartsbakterie Aktinobakterier Actinobacteria Actinomycetes oxidativ stress peroxid stress strukturbestämning karaktärisering stabilisering peroxidsensor järnsensor mangansensor metalljonshomeostas metalljoner prokaryot Structural Biology Strukturbiologi

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