In order to produce predictable and robust systems forprotein purification and detection, well characterized, small,folded domains descending from bacterial receptors have beenused. These bacterial receptors, staphylococcal protein A (SPA)and streptococcal protein G (SPG), possess high affinity to IgGand / or HSA. They are composed of repetitive units in whicheach one binds the ligand independently. The domains foldindependently and are very stable. Since the domains also havewellknown three-dimensional structures and do not containcysteine residues, they are very suitable as frameworks forfurther protein engineering. Streptococcal protein G (SPG) is a multidomain proteinpresent on the cell surface ofStreptococcus. X-ray crystallography has been used todetermine the binding site of the Ig-binding domain. In thisthesis the region responsible for the HSA affinity of ABD3 hasbeen determined by directed mutagenesis followed by functionaland structural analysis. The analysis shows that the HSAbindinginvolves residues mainly in the second α-helix. Most protein-based affinity chromatography media are verysensitive towards alkaline treatment, which is the preferredmethod for regeneration and removal of contaminants from thepurification devices in industrial applications. Here, aprotein engineering strategy has been used to improve thetolerance to alkaline conditions of different domains fromprotein G, ABD3 and C2. Amino acids known to be susceptibletowards high pH were substituted for less alkali susceptibleresidues. The new, engineered variants of C2 and ABD shownhigher stability towards alkaline pH. Also, very important forthe potential use as affinity ligands, these mutated variantsretained the secondary structure and the affinity to HSA andIgG, respectively. Moreover, dimerization was performed toinvestigate whether a higher binding capacity could be obtainedby multivalency. For ABD, binding studies showed that divalentligands coupled using non-directed chemistry demonstrated anincreased molar binding capacity compared to monovalentligands. In contrast, equal molar binding capacities wereobserved for both types of ligands when using a directed ligandcoupling chemistry involving the introduction and recruitmentof a unique C-terminal cysteine residue. The staphylococcal protein A-derived domain Z is also a wellknown and thoroughly characterized fusion partner widely usedin affinity chromatography systems. This domain is consideredto be relatively tolerant towards alkaline conditions.Nevertheless, it is desirable to further improve the stabilityin order to enable an SPA-based affinity medium to withstandeven longer exposure to the harsh conditions associated withcleaning in place (CIP) procedures. For this purpose adifferent protein engineering strategy was employed. Smallchanges in stability due to the mutations would be difficult toassess. Hence, in order to enable detection of improvementsregarding the alkaline resistance of the Z domain, a by-passmutagenesis strategy was utilized, where a mutated structurallydestabilized variant, Z(F30A) was used as a surrogateframework. All eight asparagines in the domain were exchangedone-by-one. The residues were all shown to have differentimpact on the alkaline tolerance of the domain. By exchangingasparagine 23 for a threonine we were able to remarkablyincrease the stability of the Z(F30A)-domain towards alkalineconditions. Also, when grafting the N23T mutation to the Zscaffold we were able to detect an increased tolerance towardsalkaline treatment compared to the native Z molecule. In allcases, the most sensitive asparagines were found to be locatedin the loops region. In summary, the work presented in this thesis shows theusefulness of protein engineering strategies, both to explorethe importance of different amino acids regarding stability andfunctionality and to improve the characteristics of aprotein. <b>Keywords:</b>binding, affinity, human serum albumin (HSA),albumin-binding domain (ABD), affinity chromatography,deamidation, protein A, stabilization, Z-domain, capacity,protein G, cleaning-in-place (CIP), protein engineering, C2receptor.

This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium Finegoldia magna. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule. Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.

Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and ‘off-target’ binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process. The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available. In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed. The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. / QC 20100705

<p>In order to produce predictable and robust systems forprotein purification and detection, well characterized, small,folded domains descending from bacterial receptors have beenused. These bacterial receptors, staphylococcal protein A (SPA)and streptococcal protein G (SPG), possess high affinity to IgGand / or HSA. They are composed of repetitive units in whicheach one binds the ligand independently. The domains foldindependently and are very stable. Since the domains also havewellknown three-dimensional structures and do not containcysteine residues, they are very suitable as frameworks forfurther protein engineering.</p><p>Streptococcal protein G (SPG) is a multidomain proteinpresent on the cell surface of<i>Streptococcus</i>. X-ray crystallography has been used todetermine the binding site of the Ig-binding domain. In thisthesis the region responsible for the HSA affinity of ABD3 hasbeen determined by directed mutagenesis followed by functionaland structural analysis. The analysis shows that the HSAbindinginvolves residues mainly in the second α-helix.</p><p>Most protein-based affinity chromatography media are verysensitive towards alkaline treatment, which is the preferredmethod for regeneration and removal of contaminants from thepurification devices in industrial applications. Here, aprotein engineering strategy has been used to improve thetolerance to alkaline conditions of different domains fromprotein G, ABD3 and C2. Amino acids known to be susceptibletowards high pH were substituted for less alkali susceptibleresidues. The new, engineered variants of C2 and ABD shownhigher stability towards alkaline pH. Also, very important forthe potential use as affinity ligands, these mutated variantsretained the secondary structure and the affinity to HSA andIgG, respectively. Moreover, dimerization was performed toinvestigate whether a higher binding capacity could be obtainedby multivalency. For ABD, binding studies showed that divalentligands coupled using non-directed chemistry demonstrated anincreased molar binding capacity compared to monovalentligands. In contrast, equal molar binding capacities wereobserved for both types of ligands when using a directed ligandcoupling chemistry involving the introduction and recruitmentof a unique C-terminal cysteine residue.</p><p>The staphylococcal protein A-derived domain Z is also a wellknown and thoroughly characterized fusion partner widely usedin affinity chromatography systems. This domain is consideredto be relatively tolerant towards alkaline conditions.Nevertheless, it is desirable to further improve the stabilityin order to enable an SPA-based affinity medium to withstandeven longer exposure to the harsh conditions associated withcleaning in place (CIP) procedures. For this purpose adifferent protein engineering strategy was employed. Smallchanges in stability due to the mutations would be difficult toassess. Hence, in order to enable detection of improvementsregarding the alkaline resistance of the Z domain, a by-passmutagenesis strategy was utilized, where a mutated structurallydestabilized variant, Z(F30A) was used as a surrogateframework. All eight asparagines in the domain were exchangedone-by-one. The residues were all shown to have differentimpact on the alkaline tolerance of the domain. By exchangingasparagine 23 for a threonine we were able to remarkablyincrease the stability of the Z(F30A)-domain towards alkalineconditions. Also, when grafting the N23T mutation to the Zscaffold we were able to detect an increased tolerance towardsalkaline treatment compared to the native Z molecule. In allcases, the most sensitive asparagines were found to be locatedin the loops region.</p><p>In summary, the work presented in this thesis shows theusefulness of protein engineering strategies, both to explorethe importance of different amino acids regarding stability andfunctionality and to improve the characteristics of aprotein.</p><p><b>Keywords:</b>binding, affinity, human serum albumin (HSA),albumin-binding domain (ABD), affinity chromatography,deamidation, protein A, stabilization, Z-domain, capacity,protein G, cleaning-in-place (CIP), protein engineering, C2receptor.</p>

A new Laser Induced Fluorescence Capillary Zone Electrophoresis (LIF-CZE) bioassay to detect and study the catalytic activity of the sulfur assimilating enzyme commonly found in E. coli species; alkanesulfonate monooxygenase (EC 1.14.14.5) is described for the first time. This technique enables the possibility for direct injection onto a capillary for detection without the need for pre-concentration of sample and with minimal sample preparative steps prior to analysis. In this bioassay, a group of Fischer based cyanine dyes and two Oxazine (Nile red) derivatives were designed for further optimization as key Vis-NIR fluorescent substrate. In developing this technique, the test dyes were first assessed for their photophysical properties, based on four criteria; (1) photostable (2) solvatochromism (3) binding affinity towards both the monooxygenase active site and serum albumin and (4) chemical stability in strong electric field strength. Applying key dye characterization procedures including; molar absorptivity determination, quantum yield determination, photostability, solvatochromism and protein interaction studies it was determined that the Fischer indolium cyanine dyes were most suitable for the method development. The data revealed that under the test conditions, reduced flavin, the oxidative monooxygenase catalytically specifically converts the alkylsulfonate substituted cyanine dyes to the corresponding aldehyde. This new bioassay has proven to be quick, portable, sensitive, reliable and the exhibit the possibility of ‘on-the-spot’ detection; advantages not readily realized with other commonly applied techniques such as PCR, SPR, ELISA and GC used to study bacterial sulfur assimilation processes. In addition, recent literature results proposed by other research groups developing similar techniques showed strong reliance on GC analyses. Those assays involve the use of low molecular weight straight chain non-emissive alkanesulfonate substrates. Once enzyme catalysis occurs the aldehyde is formed becomes rather volatile and requires complex and tedious headspace sampling for GC analyses. This feature limits the in vitro applicability and eliminated the possibility in vivo development. Our goal is to further develop, optimize and present this CZE based bioassay as a suitable alternative to the current trends in the field while creating a more robust and sensitive in vitro monooxygenase detection method with the possibilities of in vivo application.

The recent developments and improvements of instrumental methods for the analyses of the environmental contaminants, especially the persistent organic pollutants (POPs), have made it possible to detect and quantify these at very low concentrations in environmental and biotic matrices. The main objective of this thesis is to demonstrate the capability of the atmospheric pressure chemical ionization technique (APCI), using gas chromatography coupled to tandem mass spectrometry for the determination of a wide range of environmental contaminants, including the POPs regulated by Stockholm Convention, such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), but also the derivates of PBDEs and novel brominated flame retardants (NBFRs). The APCI was operated in charge transfer condition, preferably producing molecular ions. Multiple reaction monitoring (MRM) experiments were optimized by adjusting cone voltage, collision energy and dwell time. Optimization of source parameters, such as gas flows and temperatures was also performed. Low concentration standards were analyzed, achieving a visible chromatographic peak for 2 fg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) demonstrating the excellent sensitivity of the system. Adequate linearity and repeatability were observed for all the studied compounds. The performance of APCI methods was validated against the conventional methods using gas chromatography coupled to high resolution mass spectrometry for chlorinated compounds in a wide range of matrices including environmental, air, human and food matrices. The GC-APCI-MS/MS method was successfully applied to a set of 75 human serum samples to study the circulating levels of POPs in epidemiologic studies. Moreover the method was utilized to establish temporal trends of POPs in osprey eggs samples collected during the past five decades.

In this doctoral thesis, a novel method for the determination of primary bile acids cholic acid and chenodeoxycholic acid is presented. Bile acids play various vital roles in the mammalian body. Moreover, their determination is extremely helpful in liver and biliary disease diagnosis and management. These saturated organic compounds lack strong chromophores and fluorophores in their structure, and thus are usually hard to detect in spectroscopy. For this reason, either instrumentally advanced but expensive methods, such as mass spectrometry, or less reliable enzymatic methods are commonly employed in bile acids quantitation. Hence, the demand for simple and reliable methods for their determination is strong. Bile acids are also known to be virtually inert for direct electrochemical oxidation. Herein, a simple method for their chemical activation for electrochemical oxidation on bare electrode materials was developed, optimized and applied to cholic acid and chenodeoxycholic acid determination. The activation is based on a dehydration reaction of a primary bile acid with 0.1 mol L-1 HClO4 in acetonitrile (water content 0.55%) that introduces double bond(s) into the originally fully saturated steroid core. This naturally increases the electron density in the structure, and thus allows electrochemical...

The studies in the present thesis explored the feasibility of using nanocellulose-based filters in virus removal filtration of plasma-derived proteins.   In Paper I, two-step nanofiltration of commercially available human serum albumin (HSA) product, which was diluted to 10 g L-1 by phosphate buffer saline (PBS) and adjusted pH to 7.4, was performed to remove soluble protein aggregates and reduce filter fouling. The two-step filtration of HSA employed nanocellulose-based filters of varying thickness, i.e. 11 μm and 22 μm filters.  The removal of HSA aggregates during filtration through 11 μm pre-filters dramatically improves the flow properties of the 22 μm filter, enabling high protein throughput and high virus clearance. A distribution of pore sizes between 50 nm and 80 nm, which is present in the 11 μm filter and is absent in the 22 μm filter, plays a crucial part in removing the HSA aggregates. With respect to virus filtration, 1 bar constant trans-membrane pressure filtration shows poor removal ability of ΦX174 bacteriophage (28 nm), i.e., log10 reduction value (LRV) ≤ 3.75, while that at 3 bar and 5 bar achieves LRV[MOU1] [LW2]  &gt; 5 model virus clearance and overall rapid filtration. Removal of protein aggregates during bioprocessing of HSA products is key to improving the filtration flux, which makes it possible to apply virus removal filtration for HSA to ensure its virus safety.   In Paper II, nanofiltration of human plasma-derived intravenous immuno-globulin (IVIG) intermediate (11.26 g L-1, pH 4.9) was carried out to demonstrate high product recovery and high model virus clearance. Virus removal filtration of industrial-grade human IVIG was achieved using 33μm filters at both low (60 Lm-2) and high (288 Lm-2) volumetric load. No changes in IVIG structure were detected and high product recovery was recorded. High virus clearance (LRV ≥ 5-6) was achieved for the small-size model viruses (ΦX174 and MS2 bacteriophages) during the load volume of 60 Lm-2. Side-by-side comparisons with commercial virus removal filters suggest that the nanocellulose-based filter paper presents great potential for industrial bioprocessing of plasma-derived IVIG.   In Paper III, process analytical technology (PAT) approach was employed to identify the critical filter parameters, e.g. thickness, basis weight, pore size, and flux, affecting model virus removal efficiency using filters produced by different hot presses.  The quality parameters were analyzed with ANOVA and Shewhart charts. Compared with other studied parameters, the hydraulic flux appears as the most relevant final product quality attribute of the nanocellulose-based filter paper to reflect the virus removal efficiency. In particular, a 15% higher flux may be associated with a 0.5-1.0 log10 reduced virus clearance (p=0.007). The results are highlight the importance of continued systematic studies in quality assurance using statistical process control tools  [MOU1]Define LRV  [LW2]Defined in the line above

Prostate cancer is one of the most common types of cancer worldwide and claims hundreds of thousands of lives annually. Currently the most common treatment for prostate cancer is external beam radiotherapy, however, this treatment comes with serious side effects since it lacks selectivity for the cancer cells. Therefore, less harmful treatments are needed and sought for, such as targeted treatments that are intended to only affect cancer cells and thereby reduce the side effects. Targeted treatments require a target that differentiates the cancer cells from healthy cells. A promising target candidate that has gained attention in recent years is gastrin releasing peptide receptor (GRPR), a protein commonly overexpressed in prostate cancer cells. Furthermore, a targeting molecule intended to bind to the target is also required. For this purpose, the bombesin analogue RM26, a high affinity GRPR binder, shows promise. Previous studies have led to the development of RM26-conjugates for the purpose of targeted prostate cancer radiotherapy. In these conjugates RM26 has been linked to a DOTA-chelator for radiolabeling, and an albumin binding domain (ABD) to prolong the conjugate’s half-life in vivo by binding to human serum albumin (HSA). The idea is that the RM26-conjugate will bind to both HSA in the blood and to GRPR on the prostate cancer cells and eliminate the cancer cells with the radiation from the radionuclide attached to the DOTA-chelator. Although these earlier studied conjugates have been very promising some improvements of certain aspects need to be achieved, mainly to improve the biodistribution with retained GRPR binding affinity. Therefor the purpose of this project was to produce three new versions of previous RM26- conjugates and evaluate if they are suitable for further prostate cancer therapy studies. The three RM26-conjugates were developed with primarily recombinant expression in E. coli cells and solid phase peptide synthesis (SPPS). The characterization phase in this project was carried out with mainly five different methods: matrix-assisted laser desorption ionization time- of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization- mass spectrometry (ESI-MS), circular dichroism (CD), surface plasmon resonance (SPR) and flow cytometry. The results showed that all three new RM26-conjugates were possible to produce and yielded final products corresponding to the expected molecular weights. Furthermore, the results indicate that all three RM26-conjuagtes are stable and maintain their structural properties under in vivo- temperatures and that they have high binding affinity for HSA. Further studies need to be conducted before drawing any certain conclusions regarding GRPR binding affinity. / Prostatacancer är en av de mest vanligt förekommande cancertyperna världen över och skördar hundratusentals liv årligen. I nuläget är extern strålbehandling det vanligaste terapialternativet mot prostatacancer, men denna behandling kommer med allvarliga biverkningar på grund av att den saknar selektivitet för cancerceller. Därför finns ett stort behov av mindre skadliga behandlingsformer, såsom riktade behandlingar som endast är avsedda att påverka cancerceller och därigenom minska biverkningarna. Riktade behandlingar kräver ett mål som skiljer cancercellerna från friska celler. En lovande målkandidat som har uppmärksammats de senaste åren är gastrinfrisättande peptidreceptor (GRPR), ett protein som vanligtvis överuttrycks i prostatacancerceller. I tillägg så krävs också en målsökande molekyl avsedd att binda till målet. För detta ändamål visar bombesinanalogen RM26, en GRPR-bindare med hög affinitet, sig vara lovande. Tidigare studier har utvecklat RM26-konjugat för målinriktad strålbehandling av prostatacancer. Dessa konjugat består av en RM26-peptid bunden till en DOTA-kelator för radioinmärkning och en albuminbindande domän (ABD) för att förlänga konjugatens halveringstid in vivo genom att binda till humant serumalbumin (HSA). Syftet med RM26- konjugaten är att de ska binda till både HSA i blodet och GRPR på prostatacancercellerna, och därmed eliminera cancercellerna med strålning från den radioinmärkta DOTA-kelatorn. Även om de tidigare RM26-konjugaten har varit mycket lovande krävs det att vissa förbättringar av några aspekter uppnås, främst affiniteten för GRPR. Syftet med detta projekt var därför att producera tre nya versioner av tidigare RM26-konjugat och utvärdera ifall de uppvisar tillfredsställande egenskaper. De tre RM26-konjugaten utvecklades primärt rekombinant i E. coli-celler och fastfas- peptidsyntes (SPPS). Karaktäriseringsfasen i detta projekt genomfördes med huvudsakligen fem olika metoder: MALDI-TOF-MS, elektrosprejjonisering-masspektrometri (ESI-MS), cirkulär dikroism (CD), ytplasmonresonans (SPR) och flödescytometri. Resultaten visade att alla tre nya RM26-konjugat var möjliga att producera och gav slutprodukter motsvarande de förväntade molekylvikterna. Vidare indikerar resultaten att alla tre RM26-konjugat är stabila och bibehåller sina strukturella egenskaper under in vivo-temperaturer och att de har hög affinitet för HSA. Ytterligare studier bör utföras innan säkrare slutsatser kan dras angående GRPR-bindningsaffinitet.

Marknaden för proteinbaserade läkemedel, de så kallade biologiska läkemedlen, är idag en industri som omsätter miljarder. Ett vanligt sätt att utveckla dessa läkemedel på är med hjälp av monoklonala antikroppar då de kan binda till sitt mål med hög specificitet. Däremot begränsas denna teknik av en lång och dyr produktion som dessutom kräver däggdjursbaserade uttrycksystem. En alternativ teknik till de monoklonala antikropparna är att använda små proteiner som enkelt kan produceras i bakterier till en låg kostnad. Dock begränsas denna metod av de små proteinernas korta cirkuleringstid i blodet. I ett tidigare projekt, har ett litet protein vid namnet ABDderived affinity ProTein (ADAPT) på cirka 7 kDa, utvecklats för att kunna binda till både humant serumalbumin (HSA) för att förlänga cirkulationstiden i blodet och Interleukin 17c (IL17c) som är ett pro-inflammatorisk cytokin. Studien visade dock att ADAPT proteinet inte samtidigt kunde binda till de båda molekylerna tillräckligt effektivt. Syftet med denna uppsats är därför att undersöka om det nämnda proteinet kan optimeras genom så kallad multimering och/eller manipulering av bindningssätet för HSA i syfte att åstadkomma en effektiv och mer långvarig cirkulationstid i blodet samtidigt som det binder sig till sitt mål, IL17c. Tio nya versioner av ADAPT proteinet har utvecklats genom att klona och transformera proteiner till en högt producerande Escherichia coli (E. coli) stam. Proteinerna har sedan producerats och renats fram. Det kunde observeras att proteinerna hade den önskade renheten för att kunna karaktäriseras. Vidare var det möjligt att se att proteinerna hade sin önskade molekylvikt och erhöll sin förväntade struktur som en alfahelix. Proteinernas smältpunkter hade förbättrats eller var liknande jämfört med det ursprungliga proteinet. Dessutom kunde alla proteiner återgå till sin ursprungliga struktur efter upphettning. Utvärderingen av proteinernas bindningskapacitet, med original proteinet som referens, visade på en ökad affinitet till sitt mål, IL17c, för två dimerer och trimeren samt en jämförbar affinitet för två av monomererna med ett manipulerat bindingssäte till HSA. Interaktion till HSA var jämförbar med den ursprungliga ADAPT molekylen för alla nya varianter förutom monomererna med ett manipulerat bindingssäte och dimeren med två manipulerat bindingssäten till HSA. Evaluering av de nya proteinernas kapacitet att binda samtidigt till HSA och IL17c visade att det var gynnsamt med en dimereiserad molekyl då det skapade en distans mellan molekylerna och dess bindningssäten. Vidare kunde det också visas att ordningen som molekylerna interagerade med varandra påverkade proteinernas simultana bindning. / The market for protein-based drugs, or the so-called biopharmaceuticals, is a multibillion-dollar industry today. In the development of protein-based drugs it is common to use monoclonal antibodies (mAbs) due to their ability to bind to its target with high specificity. However, therapeutical development of mAbs is limited by its long and expensive production in mammalian expression system. An alternative to mAbs are the so-called alternative scaffolds which are small proteins that can be produced in bacteria at lower costs. Although a drawback with the latter proteins is their short serum half-life. A small scaffold protein, ABD-Derived Affinity ProTein (ADAPT) of approximate 7 kDa was earlier engineered to obtain bispecific affinity, to Human Serum Albumin (HSA), to extend its half-life, as well as to the pro-inflammatory cytokine, Interleukin 17c (IL17c). Unfortunately, it was shown that the simultaneous binding was not efficient enough for its desired purpose. The aim with this project was therefore to investigate if the previous mentioned binder could be optimized by multimerization and/or manipulation of the HSA binding site for an efficient half-life extension. By generating ten new designs of the ADAPT variants, it was observed that the new variants had stable alpha helical structures and an improved or similar melting temperature as the original variant. The evaluation of the target binding displayed an improved affinity to the target, IL17c, for two of the dimeric versions as well as for the trimer and a comparable affinity for two of the monomers with a manipulated HAS binding site. The interaction to HSA was comparable to the original ADAPT for all binders except from the monomers with impaired HSA binding and the dimer with two impaired HSA binding sites. The evaluation of the simultaneous binding showed that it was favored by dimerization when a distance between the two molecule and their binding surfaces was added. Moreover, it could also be seen that the order of binding events had an impact on the simultaneous binding.