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.

Proteiner spelar en avgörande roll i människokroppen och det är därför viktigt att inta tillräckligt med essentiella aminosyror genom kosten. Tidigare har animaliska källor varit kända för att vara rika på dessa aminosyror, men nu förtiden räcker det inte för att tillfredsställa den globala behov. Dessutom väcker den animaliska proteiner allvarliga frågor om klimatförändringar och etik. Ärtor innehåller en essentiell profil av aminosyror och har många andra fördelar, såsom tillgänglighet, kostnadseffektivitet och förmåga att växa i låga temperaturer. Tidigare studier har visat att regelbunden intag av ärtprotein kan minska risken för hjärt-kärlsjukdomar och diabetes. Studier visade att Naturliga djupa eutektiska lösningsmedel (NADES) kan lösa upp proteiner utan att denaturera de, vilket är ett problem med traditionella lösningsmedel som kan påverka proteinstrukturer. Dessutom är NADES icke-toxiska och biologiskt nedbrytbara lösningar, vilket gör dem mer hållbara och miljövänliga än vanliga vätskor som används vid proteinextraktion. Syftet med studien var att använda NADES för att utveckla en ny metod för att extrahera proteiner från gula ärtor och jämföra denna med etablerade metoder. Dessutom skulle skillnader i proteinutbyte och strukturella förändringar uppskattas genom användning av Size Exclusion Chromatography (SEC) och Sodium Dodecyl Sulfate PolyAcrylamide Gel Elektrofores SDS-PAGE. Tre varianter av NADES framställdes från kolinklorid, citronsyra och olika vattenhalter. NADES 20% användes för extraktion, eftersom det har visat sig vara en lämplig metod för detta ändamål. Resultaten visade att extraktion med NADES är mer komplicerad än referensmetoderna för att proteinerna utfölls vid olika steg under extraktion process. SEC-kromatogram visade proteindenaturering där en tydlig topp erhölls vid senare elueringsvolym. SDS-PAGE analys genomfördes för att identifiera toppar men inge resultat kunde erhålla på grund av låg proteinkoncentration. Från ett optimistiskt perspektiv kan de denaturerade proteinerna fortfarande användas inom livsmedelsindustrin eller NADES kan användas som en pre-extraktionsmetod för att avlägsna föroreningar innan den verkliga extraktionsprocessen påbörjas. Det skulle vara användbart för framtida studier att prova att extrahera med en lägre vattenhalt i NADES eller med en annan typ av NADES. / Protein plays a crucial role in the human body, making it essential to consume sufficient amounts of essential amino acids through diet. Traditionally, animal sources have been known to be rich in these amino acids, but this is not sufficient to meet global demand. Additionally, this raises serious questions about climate change and ethics. Peas have been found to contain an essential profile of amino acids and have many other advantages, such as availability, cost-effectiveness, and the ability to grow in low temperatures. Previous studies have shown that regular consumption of pea protein can reduce the risk of cardiovascular disease and diabetes. Studies have shown that Natural Deep Eutectic Solvents (NADES) can dissolve proteins without denaturing them, which is a problem with traditional solvents that can affect protein structures. In addition, NADES are non-toxic and biodegradable solutions, making them more sustainable and environmentally friendly than common liquids used in protein extraction. The aim of the study was to use NADES to develop a new protein extraction method from yellow peas and compare this with established methods. In addition, differences in protein yield and structural changes were estimated using Size Exclusion Chromatography (SEC) and Sodium Dodecyl Sulfate PolyAcrylamide Gel Elektrofores SDS-PAGE. Three variants of NADES were prepared from choline chloride, citric acid, and different water contents. NADES 20% was used for extraction as it has been found to be a suitable method for this purpose. The results showed that extraction with NADES is more complicated than the reference methods because the proteins precipitated at different stages during the extraction process. SEC chromatogram showed protein denaturation where a clear peak was obtained at later elution volumes. SDS-PAGE analysis was performed to identify peaks, but no results could be obtained due to low protein concentration. From an optimistic perspective, the denatured proteins can still be used in the food industry, or NADES can be used as a pre-extraction method to remove impurities before the actual extraction process begins. It would be useful for future studies to try extracting with a lower water content in NADES or with a different type of NADES.

TRIM21 is a cytosolic ubiquitin ligase and an antibody receptor that providesa last line of defense against invading pathogens. By utilizing the diversity ofantibody repertoire to identify pathogens, TRIM21 serves as a link betweenintrinsic cellular defense and adaptive immunity. A variety of diseases havebeen linked to mutations of the TRIM family, including cancer, inflammatorydiseases, and autoimmune diseases. In this project, TRIM21 was producedand purified from Escherichia coli, (E.coli). Protein characterization wasperformed with SDS-PAGE, size exclusion chromatography and cryo-electronmicroscopy (cryo-EM). Previously TRIM21 has been shown to form a dimerwhen produced in SF9. Results from size exclusion chromatography show thatTRIM21 form a larger complex when expressed in E.coli. Cryo-EM resultsshow that the complex structure is more globular than previously thought.Purified TRIM21 was bound to the antibody IC100. SDS-PAGE and sizeexclusion chromatography results show much lower affinity to antibodies thanexpected. / TRIM21 är en cytosolisk ubiquitinligas- och antikroppsreceptor som ger ensista försvarslinje mot invaderande virus. Genom att använda mångfalden avantikroppsrepertoar för att identifiera patogener, fungerar TRIM21 som enlänk mellan inre cellulärt försvar och adaptiv immunitet. En mängd olikasjukdomar har kopplats till mutationer i TRIM-familjen, inklusive cancer,inflammatoriska sjukdomar och autoimmuna sjukdomar. I detta projekt produceradesoch renades TRIM21 från Escherichia coli, (E.coli). Proteinkarakteriseringutfördes med SDS-PAGE, gelfiltreringskromatografi och kryo-elektronmikroskopi(cryo-EM). Tidigare har TRIM21 visat sig bilda en dimer när den producerasi SF9. Resultat från gelfiltrering visar att TRIM21 bildar ett större komplexnär det uttrycks i E.coli. Cryo-EM-resultat visar att den komplexa strukturenär mer klotformig än man tidigare trott. Renad TRIM21 bands till antikroppenIC100. SDS-PAGE och gel-filtrerings resultat visar mycket lägre affinitet tillantikroppar än förväntat.

Winter rye produces ice-modifying antifreeze proteins upon cold treatment. Two of these antifreeze proteins are members of the large, highly conserved, ??-1,3-endoglucanase family. This project was designed to identify glucanase genes that are expressed during cold acclimation, wounding, pathogen infection, drought or treatment with the phytohormones ethylene and MeJa. Additionally, a more detailed proteomic analysis was to be carried out to evaluate the glucanase content of the apoplast of cold-acclimated (CA) winter rye. Results of 2D SDS-PAGE analysis revealed that non-acclimated whole leaf protein extracts contain at least two ??-1,3-endoglucanses while CA whole leaf protein extracts contain at least three ??-1,3-endoglucanses. Subsequent 2D SDS-PAGE analysis was conducted on the apoplast extracts of NA and CA winter rye plants revealed the limitations of standard 1D SDS-PAGE. The 2-dimensional gel analysis revealed that there is a minimum of 25 proteins within the apoplast of CA winter rye, including at least 5 ??-1,3-endoglucanases. Genome walking was used to isolate cold-responsive glucanase genes. The five genes isolated were designated scGlu6, scGlu9, scGlu10, scGlu11 and scGlu12. The cis-element pattern within the promoter of each gene was evaluated using online databases of documented plant cis elements. As expected, all of the promoters contained elements associated with cold, biotic and abiotic stresses, light regulation, and development. The expression patterns predicted by the cis elements in each promoter were compared to the mRNA abundance produced by each gene as detected by semi-quantitative reverse transcriptase PCR. In most cases, the abundance of transcripts arising from each gene loosely corresponded to the expression pattern predicted by the cis elements the corresponding promoter. Transcripts of scGlu9, 10 and 11 were present in cold-treated tissues and are candidates for ??-1,3-endoglucanases with antifreeze activity. The results presented in this thesis provide additional insight into the apoplast proteome of CA winter rye plants as well as the complexity of the signals controlling the proteins that reside there. Although there are still a number of unresolved questions, this research opens new directions for future studies in the cold acclimation process in winter rye and specifically for the contribution of ?? -1,3-endoglucanses.

Introduction: Cellular membrane proteins, such as the cytochrome P450 enzyme superfamily (P450), have important roles in the physiology of the cell. P450s are important in metabolising endogenous molecules, as well as metabolising xenobiotic substances for detoxification and excretion. P450s are also implicated in cancer as they can act to 'negatively' de-activate or 'positively' activate cancer therapeutics. Identifying specific P450s that are highly up-regulated at the tumour site could be used to predict drug response and formulate targeted cancer therapy to help diminish systemic side-effects. Methods: Previous enrichment strategies have been unable to isolate the full complement of the P450 superfamily. To develop enrichment procedures for the P450s, a proteomic strategy was developed so that compounds could be screened for their effectiveness as general P450 probes. A standardised work-flow was created, encompassing affinity chromatography, protein concentration/desalting, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-mass spectrometry (HPLC-MS). A ketoconazole analogue and a 2-EN analogue, with known P450 inhibition, were immobilised on a solid support for comparison to immobilised histamine. Co-factor removal, competitive elution and DTT cleavage of disulfide bonds of probes were utilised to elute bound proteins. Results/Discussion: Inhibitor-beads bound a large range of proteins, including P450's, of which some were eluted by co-factor removal, some by competitive elution. Specificity of binding was improved by optimising buffer conditions and solid supports, however non-specific binding was not totally eradicated. All human P450s from spiked samples and 18 P450s from more complex mouse liver samples were recovered using one or more ligands.

<p>There is convincing research which shows that the enzyme alkaline phosphatase (ALP) has a central role in the mineralization of bone, more precisely that its catalytic activity is needed in the process. ALP is found on the surface of matrix vesicles where the mineral is formed. One theory about the function of the enzyme is that it binds to fibrous collagen in the bone and thereby incorporating the mineral into the bone. The purpose of this study is to establish whether ALP binds to collagen. If this is the case, more elaborate studies around this will be performed. The strength of the binding between collagen and the different types of ALP will be evaluated, as well as on which part of the collagen the binding occurs. The binding is going to be studied by constructing a method for the ÄKTApurifier system.</p><p> </p><p>Initially, the pureness of the different type of collagens was determined by using SDS-PAGE and the activity of the different types of ALP was established. These were also compared with a native PAGE. In SDS-PAGE, bovine type I collagen showed markings for a triple helix, a double helix and two single strains, α₁ and α₂. Bovine type II collagen showed markings for a double helix and α₁-strains. Human type I collagen showed markings for a triple helix, two double helixes, two α-strains and contaminations. Trials with collagen in Native PAGE did not provide any results. However, the trials with ALP revealed that the different types of ALP had different charge.</p><p> </p><p>Thereafter, blotting was performed. The results showed that all the different types of ALP, besides from E. coli, binds to bovine collagen type I and II and human collagen type I, however within various periods of time. In the trials with collagen coated plates the acquired results showed that some of the different types of ALP bind to collagen. ALP from liver binds the strongest to both collagen type I from rat and type IV from mouse. Intestinal ALP also binds to both types of collagen but not nearly as strong as liver ALP. Serum from rats did bind to collagen type I from rat but not to collagen type IV from mouse. ALP from kidney and human serum did not bind to either types of collagen. The trials concerning the ÄKTApurifier system were executed with ALP from liver alone because it had been proven to bind to bovine type I collagen through the previous methods. The results confirmed that ALP from liver binds to this type of collagen.</p><p> </p><p>The conclusions from this study are that ALP does indeed bind to collagen and does so to the triple helix and double helix form as well as the single strains of collagen. In other words the part of the structure in collagen that ALP binds to must exist in all three stages of collagen formation. Furthermore, it seems like some of the different types of ALP has a higher affinity for binding to collagen, as the time for binding to collagen varies for the different types of ALP. The results differed between methods concerning different types of ALP. Although, the method we consider to give the best result was blotting. However, the method using ÄKTApurifier can be complementary but needs further development.</p>

<p>Fotosyntesen kallas den process som omvandlar ljusenergi till kemisk energi. Fotosyntesen sker i tylakoidmembranet och drivs av två stora proteinkomplex, fotosystem II (PSII) och fotosystem I (PSI) då de tillförs energi i form av fotoner. PSI-N är ett mindre protein på ca 10 kDa som ingår i PSI. På något sätt, som ännu inte är klarlagt, samverkar PSI-N med PSI-F och plastocyanin när det dockar till PSI. Det är därför av viktigt att rena fram större mängder av PSI-N för att få djupare kunskaper om proteinet samt dess struktur och funktioner. Tidigare undersökningar har utförts i ämnet och ett fusionsprotein innehållande PSI-N har uttryckts i <em>Escherichia coli</em> (<em>E.coli</em>). Problem har dock uppstått efter lysis av cellerna då det har visat sig att fusionsproteinet har degraderats. Vårt examensarbete strävar efter att rena fram intakt fusionsprotein med hjälp av, framför allt, mekanisk lysis och proteasinhibitorer.</p> / <p>The process where light is converted into chemical energy is called photosyntesis. The reaction takes place in the thylakoid membrane and is driven by two major protein complexes, photosystem II (PSII) and photosystem I (PSI) when energy in form of photons are received. PSI-N, a subunit in PSI, is a smaller protein with a mass of approximately 10 kDa. In some way, which is not yet clarified, PSI-N collaborates with PSI-F and plastocyanin when plastocyanin is docking to PSI. It is therefore important to purify larger amounts of the protein to acquire deeper knowledge of its structure and function. In earlier research the PSI-N protein has been expressed in <em>Escherichia coli</em> (<em>E.coli</em>). The problem has been degradation of the fusion protein after lysis. Our goal with this project is to obtain the purified protein intact using mechanic lysis and protease inhibitors.</p>

There is convincing research which shows that the enzyme alkaline phosphatase (ALP) has a central role in the mineralization of bone, more precisely that its catalytic activity is needed in the process. ALP is found on the surface of matrix vesicles where the mineral is formed. One theory about the function of the enzyme is that it binds to fibrous collagen in the bone and thereby incorporating the mineral into the bone. The purpose of this study is to establish whether ALP binds to collagen. If this is the case, more elaborate studies around this will be performed. The strength of the binding between collagen and the different types of ALP will be evaluated, as well as on which part of the collagen the binding occurs. The binding is going to be studied by constructing a method for the ÄKTApurifier system.   Initially, the pureness of the different type of collagens was determined by using SDS-PAGE and the activity of the different types of ALP was established. These were also compared with a native PAGE. In SDS-PAGE, bovine type I collagen showed markings for a triple helix, a double helix and two single strains, α1 and α2. Bovine type II collagen showed markings for a double helix and α1-strains. Human type I collagen showed markings for a triple helix, two double helixes, two α-strains and contaminations. Trials with collagen in Native PAGE did not provide any results. However, the trials with ALP revealed that the different types of ALP had different charge.   Thereafter, blotting was performed. The results showed that all the different types of ALP, besides from E. coli, binds to bovine collagen type I and II and human collagen type I, however within various periods of time. In the trials with collagen coated plates the acquired results showed that some of the different types of ALP bind to collagen. ALP from liver binds the strongest to both collagen type I from rat and type IV from mouse. Intestinal ALP also binds to both types of collagen but not nearly as strong as liver ALP. Serum from rats did bind to collagen type I from rat but not to collagen type IV from mouse. ALP from kidney and human serum did not bind to either types of collagen. The trials concerning the ÄKTApurifier system were executed with ALP from liver alone because it had been proven to bind to bovine type I collagen through the previous methods. The results confirmed that ALP from liver binds to this type of collagen.   The conclusions from this study are that ALP does indeed bind to collagen and does so to the triple helix and double helix form as well as the single strains of collagen. In other words the part of the structure in collagen that ALP binds to must exist in all three stages of collagen formation. Furthermore, it seems like some of the different types of ALP has a higher affinity for binding to collagen, as the time for binding to collagen varies for the different types of ALP. The results differed between methods concerning different types of ALP. Although, the method we consider to give the best result was blotting. However, the method using ÄKTApurifier can be complementary but needs further development.

In this study, a new bacteriocin isolated from a Leuconostoc mesenteroides subsp. cremoris strain was purified to homogeneity by pH mediated cell adsorption-desorption, solid phase extraction with Amberlite XAD-16, cation-exchange chromatography and hydrophobic interaction chromatography. The purification resulted in an electrophoretically pure protein that was smaller than 6 kDa as judged by SDS-PAGE. The bacteriocin was found to be very hydrophobic and cationic and could be adsorbed by synthetic calcium silicate due to its cationic and especially hydrophobic nature. It was observed that this bacteriocin was sensitive to &amp / #945 / -amylase in addition to proteinase K, trypsine, pepsine and chymotrypsine and had a bactericidal mode of action with a concomitant cell lysis. The results indicated that bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was more stable to combined effect of pH and heat than nisin, lacticin 481, lacticin 3147 and lactococcin G and was bactericidal between pH 2.0-12. It was found that the bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was stable to organic solvents and could be extracted with chloroform containing solvents efficiently for purification. The bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was found to have a strong inhibitory activity against Listeria innoqua, Listeria monocytogenes, Bacillus cereus, Enterococcus faecalis, Lactobacillus delbrueckii, Lactobacillus cremoris, other Leuconostoc meenteroides strains and gram-negative bacterium Pseudomonas fluorescens. Some of the insensitive bacteria were observed to be sensitive when high concentration of the bacteriocin was used.

In recent years, biocatalytical steps in chemical synthesis are becoming increasingly important for economical and environmental-friendly production. In order to evaluate the use of enzymes in a process at Cambrex Karlskoga AB, an expression system was developed for a dehydrogenase. A synthetic gene was cloned into Escherichia coli DH5a cells, using the pTZ19R expression vector, as previously described in the literature. Protein expression was carried out at 25°C, 30°C and 37°C and results were measured using SDS-PAGE and activity assays. To improve expression, the gene was modified in three ways using PCR, yielding eight clones: It was inserted into the pSE420 expression vector, shortened to avoid inclusion body formation and a missing nucleotide was inserted into the sequence. A protocol for inclusion body screening was also developed. Finally, an assay for determining the kinetic constants of dehydrogenase was designed. It is concluded that further experiments must be done to obtain expression of the dehydrogenase and recommendations for additional work are given. / Biokatalytiska processteg har de senaste åren blivit ett allt viktigare inslag i kemisk syntes för att åstadkomma ekonomisk och miljövänlig produktion. För att utvärdera användandet av enzymer i en process hos Cambrex Karlskoga AB utvecklades ett expressionssystem för ett dehydrogenas. En syntetisk gen klonades in i Escherichia coli DH5a och uttrycktes med hjälp av expressionsvektorn pTZ19R, som tidigare finns beskrivet i litteraturen. Proteinuttrycket utfördes vid 25°C, 30°C och 37°C och resultatet mättes med hjälp av SDS-PAGE och aktivitetsmätningar. Genen för dehydrogenaset modifierades på tre sätt, vilket gav upphov till åtta varianter. Genen fördes över till expressionsvektorn pSE420, kortades för att undvika bildning av inklusionskroppar och en nukleotid som fattades från gensekvensen återinfördes. Ett protokoll utarbetades även för undersökning av inklusionskroppar. Till sist sammanställdes en metod för att undersöka de kinetiska konstanterna hos dehydrogenaset. Slutsatsen av arbetet är att fortsatta studier måste utföras för att erhålla uttryck av dehydrogenaset och rekommendationer ges för framtida undersökningar.