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.

Fragment-based drug discovery is an emerging process that has gained popularity in recent years. The process starts from small molecules called fragments. One major step in fragment-based drug discovery is fragment screening, which is a strategy to screen libraries of small molecules to find hits. The strategy in theory is more efficient than traditional high-throughput screening that works with larger molecules. As fragments intrinsically possess weak affinity to a target, detection techniques of high sensitivity to affinity are required for fragment screening. Furthermore, the use of different screening methods is necessary to improve the likelihood of success in finding suitable fragments. Since no single method can work for all types of screening, there is a demand for new techniques. The aim of this thesis is to introduce weak affinity chromatography (WAC) as a novel technique for fragment screening. WAC is, as the name suggests, an affinity-based liquid chromatographic technique that separates compounds based on their different weak affinities to an immobilized target. The higher affinity a compound has towards the target, the longer it remains in the separation unit, and this will be expressed as a longer retention time. The affinity measure and ranking of affinity can be achieved by processing the obtained retention times of analyzed compounds. In this thesis, WAC is studied for fragment screening on two platforms. The first system comprised a 24-channel affinity cartridge that works in cooperation with an eight-needle autosampler and 24 parallel UV detector units. The second system was a standard analytical LC-MS platform that is connected to an affinity column, generally called WAC-MS or affinity LC-MS. The evaluation criteria in studying WAC for fragment screening using these platforms were throughput, affinity determination and ranking, specificity, operational platform characteristics and consumption of target protein and sample. The model target proteins were bovine serum albumin for the first platform, thrombin and trypsin for the latter. Screened fragments were either small molecule drugs, a thrombin-directed collection of compounds, or a general-purpose fragment library. To evaluate WAC for early stages of fragment elaboration, diastereomeric mixtures from a thrombin-directed synthesis project were screened. Although both analytical platforms can be used for fragment screening, WAC-MS shows more useful features due to easy access to the screening platform, higher throughput and ability to analyze mixtures. Affinity data from WAC are in good correlation with IC50 values from enzyme assay experiments. The possibility to distinguish specific from non- specific interactions plays an important role in the interpretation of WAC results. In this thesis, this was achieved by inhibiting the active site of the target protein to measure off-site interactions. WAC proves to be a sensitive, robust, moderate in cost and easy to access technique for fragment screening, and can also be useful in the early stages of fragment evolution. In conclusion, this thesis has demonstrated the proof of principle of using WAC as a new tool to monitor affinity and to select hits in fragment-based drug discovery. This thesis has indicated the primary possibilities, advantages as well as the limitations of WAC in fragment screening procedures.  In the future, WAC should be evaluated on other targets and fragment libraries in order to realize more fully the potential of the technology.

<p>Recombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated.</p><p>Attempts to create general production strategies forrecombinant protein immunogens in such a way that these areadapted for association with an adjuvant formulation wereevaluated. Different hydrophobic amino acid sequences, beingeither theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene leveleither N-terminally or C-terminally to allow association withiscoms. In addition, affinity tags derived from<i>Staphylococcus aureus</i>protein A (SpA) or streptococcalprotein G (SpG), were incorporated to allow efficient recoveryby means of affinity chromatography. A malaria peptide, M5,derived from the central repeat region of the<i>Plasmodium falciparum</i>blood-stage antigen Pf155/RESA,served as model immunogen in these studies. Furthermore,strategies for<i>in vivo</i>or<i>in vitro</i>lipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogendeltaSAG1 derived from<i>Toxoplasma gondii</i>. Both strategies were found to befunctional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms werefurthermore capable of inducing antigen-specific antibodyresponses upon immunization of mice, and we thus believe thatthe presented strategies offer convenient methods for adjuvantassociation.</p><p>Recombinant production of a respiratory syncytial virus(RSV) candidate vaccine, BBG2Na, in baby hamster kidney(BHK-21) cells was investigated. Semliki Forest virus(SFV)-based expression vectors encoding both intracellular andsecreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved bycombining serum-free production with a recovery strategy usinga product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G proteinpart of the product.</p><p>Plasmid vectors encoding cytoplasmic or secreted variants ofBBG2Na, and employing the SFV replicase for self-amplification,was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms ofBBG2Na expression and localization. Upon intramuscularimmunization of mice, the plasmid vector encoding the secretedvariant of the antigen elicited significant anti-BBG2Na titersand demonstrated lung protective efficacy in mice. This studyclearly demonstrate that protective immune responses to RSV canbe elicited in mice by DNA immunization, and that differentialtargeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protectiveefficacy.</p><p>We further evaluated DNA and RNA constructs based on the SFVreplicon in comparison with a conventional DNA plasmid forinduction of antibody responses against the<i>P. falciparum</i>Pf332-derived antigen EB200. In general,the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNAplasmid. Also recombinant SFV suicide particles inducedEB200-reactive antibodies. Importantly, all immunogens inducedan immunological memory, which could be efficiently activatedby a booster injection with EB200 protein.</p><p><b>Keywords</b>: Affibody, Affinity chromatography, Affinitypurification, DNA immunization, Expression plasmid, Fusionprotein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria,Mammalian cell expression, Recombinant immunogen, RespiratorySyncytial Virus, Semliki Forest virus, Serum albumin,<i>Staphylococcus aureus</i>protein A, Subunit vaccine,<i>Toxoplasma gondii</i></p>

<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>

The contribution of Cys17 and surface-exposed histidine residues in rhG-CSF interaction with Cu(II), Ni(II) and Hg(II) ions chelated by Light Resistant Yellow 2KT-polyethylene glycol derivative was evaluated in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran. It was determined that His43, His52, His156 and His170 residues are involved in protein interaction with chelated Cu(II) ions. Protein interaction with chelated Ni(II) is governed by His52 and His170 residues, though Cys17 is also involved. The contribution of Cys17 side chain is dominant in the interaction between rhG-CSF and chelated Hg(II) ions. The direct interaction between chelated Hg(II) ions and the –SH group of protein was determined for the first time. Based on the study of the interaction between rhG-CSF and chelated metal ions, rhG-CSF was successfully refolded from inclusion bodies in aqueous two-phase systems PEG-dextran containing chelated Ni(II) or Hg(II) ions for the first time. The refolding of rhG-CSF (C17S) in these systems was more effective compared to that of intact rhG-CSF. The dependence of refolding efficiency of rhG-CSF (C17S) in two-phase systems containing chelated metal ions on the number of histidine mutations was evaluated. It was determined that the refolding efficiency of protein in the systems containing chelated Ni(II) is inversely proportional to the number of histidine mutations. The affinity of purified rhG-CSF (C17S) and its histidine mutants for... [to full text]

Various methods for extraction and analysis of high value minor proteins (lactoferrin, lactoperoxidase and immunoglobulins) directly from raw milk were explored. Extraction, purification and analysis of high-value minor proteins directly from milk without pre-treatment are major challenges for dairy industry, largely due to the complexity of milk and the presence of colloidal solids (casein micelles and milk fat globules). To overcome some of these challenges, this work focused on three main objectives: 1) characterization of cryogel monolith chromatography for purification of lactoferrin (LF) and lactoperoxidase (LP) directly from raw milk in single step, 2) identification and characterization of Protein A Mimetic affinity ligands for purification of immunoglobulins (Igs) from milk and 3) development and validation of a surface plasmon resonance method for simultaneous quantification of five whey proteins in multiple samples. Results portrayed the possibility of 40–50 column volumes of various milk samples (whole milk, skim milk and acid whey) to pass through a 5 mL cryogel monolith chromatography column at 525 cm hr⁻¹ without exceeding its pressure limits if the processing temperature is maintained around 35–37°C. Ideally, this should be the milk secretion temperature. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg mL⁻¹) was similar to that of the binding capacity (2.01 mg mL⁻¹) at equilibrium with 0.1 mg mL⁻¹ of lactoferrin in the feed samples. Lactoferrin and lactoperoxidase was selectively bound to the cryogel column with trivial leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of 85% and a purity of 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed-bed chromatography for direct capture of proteins from milk, provided that the binding capacity can be increased. A Protein A Mimetic (PAM) hexapeptide (HWRGWV) peptide ligand that binds to the Fc portion of antibody molecules was explored for affinity purification of immunoglobulins from milk. The peptide has the ability to purify IgG from various milk and whey samples with a purity of greater than 85% in single step. More than 90% bound IgG was recovered with 0.2 M acetate buffer at pH 4.0 and total column regeneration was successfully achieved by 2.0 M guanidine-HCl. At 9.0 mg mL⁻¹ of IgG feed concentration, an equilibrium binding capacity of 21.7 mg mL⁻¹ and dynamic binding capacity of approximately 12.0 mg mL⁻¹ of resin was obtained. Recoveries and yields of IgG were significantly influenced by the feed IgG concentration. PAM hexamer ligand also contributed a significant amount of cross-reactivity with casein, glycomacropeptides and β-lactoglobulin proteins, however majority of these proteins were recovered in the regeneration step, except β-lactoglobulin, which co-eluted with IgG. Higher IgG concentration in feed vastly reduced the amount of cross-reactivity whilst increasing the recoveries and purities in the final product. PAM affinity ligands also showed interactions towards other classes of bovine immunoglobulins. These findings established the possibility of using PAM hexamer peptide as an alternative to conventional Protein A/G affinity chromatography for the isolation of Igs from milk in single step process. A surface plasmon resonance (SPR) method was developed for simultaneous, quantitative determination of commercially important whey proteins in raw and processed milk samples, whey fractions and various milk-derived products, with six samples per assay. Immobilized antibody stability and reproducibility of analyses were studied over time for 25 independent runs (n=300), giving a relative standard deviation (RSD) of <4%. Immobilized antibodies showed negligible non-specific interactions (<2–4 SPR response units (RU)) and no cross-reactivity towards other milk components (<1 RU). Regeneration of immobilised antibodies with glycine at pH 1.75 was determined to be optimal for maintaining the SPR response between samples. This method compared and validated well with reversed phase high performance liquid chromatography (RP-HPLC) and standard enzyme-linked immunosorbent assays (ELISA).

A virus-like particle (VLP) is a biological nanoparticle. It consists of the protective protein shell of a virus that is devoid of the nucleic acid required for viral replication. VLPs have two key uses: they can act as vaccines by inducing an immune response similar to their native virions, or they can facilitate gene therapy and drug delivery by encapsulating non-viral molecules and efficiently transporting them into cells. Manufacture of VLPs involves cell-based expression of virus-shell protein, with particle assembly and purification following one of two paradigms: (i) in vivo VLP assembly, followed by purification of full particles from cell lysate; (ii) partially assembled protein is recovered from cell lysate and assembled into VLPs in vitro. The flexibility and efficiency of both of these VLP manufacturing paradigms can be improved by first gaining a fundamental understanding of what is happening at key process steps. These improvements will lower the cost of VLP manufacture and enhance the viability of VLP products in the biopharmaceutical marketplace. The research reported here yielded positive outcomes for two key steps of the VLP manufacturing process, using murine polyomavirus VLPs for all experimentation. Firstly, enhanced understanding concerning the capture of virus shell protein in pentamer form (capsomeres) from cell lysate using glutathione-S-transferase (GST) affinity chromatography was obtained. It was discovered that prokaryotic expression of GST-tagged capsomeres yielded soluble aggregates having variable size distribution. Methods were developed to physically and chemically characterise these soluble aggregates, and the mechanism by which they adsorb to the chromatography resin was described using an established mathematical model. Secondly, particle characterisation of whole VLPs isolated from cell lysate was undertaken. Methods utilizing three orthogonal and quantitative techniques were developed to suggest that encapsulation of non-viral molecules (nucleic acids or proteins) during in vivo assembly causes distinct changes to the size distribution of isolated VLPs: transmission electron microscopy (TEM), asymmetrical flow field-flow fractionation with multiple-angle light scattering (AFFFF-MALS) and electrospray differential mobility analysis (ES-DMA). The understanding gained from the research presented in this work enables the enhanced capture of partially assembled virus shell protein from cell lysate, as well as a method to efficiently and cost-effectively analyse VLP solutions for the presence of desirable or undesirable encapsulated material.

A virus-like particle (VLP) is a biological nanoparticle. It consists of the protective protein shell of a virus that is devoid of the nucleic acid required for viral replication. VLPs have two key uses: they can act as vaccines by inducing an immune response similar to their native virions, or they can facilitate gene therapy and drug delivery by encapsulating non-viral molecules and efficiently transporting them into cells. Manufacture of VLPs involves cell-based expression of virus-shell protein, with particle assembly and purification following one of two paradigms: (i) in vivo VLP assembly, followed by purification of full particles from cell lysate; (ii) partially assembled protein is recovered from cell lysate and assembled into VLPs in vitro. The flexibility and efficiency of both of these VLP manufacturing paradigms can be improved by first gaining a fundamental understanding of what is happening at key process steps. These improvements will lower the cost of VLP manufacture and enhance the viability of VLP products in the biopharmaceutical marketplace. The research reported here yielded positive outcomes for two key steps of the VLP manufacturing process, using murine polyomavirus VLPs for all experimentation. Firstly, enhanced understanding concerning the capture of virus shell protein in pentamer form (capsomeres) from cell lysate using glutathione-S-transferase (GST) affinity chromatography was obtained. It was discovered that prokaryotic expression of GST-tagged capsomeres yielded soluble aggregates having variable size distribution. Methods were developed to physically and chemically characterise these soluble aggregates, and the mechanism by which they adsorb to the chromatography resin was described using an established mathematical model. Secondly, particle characterisation of whole VLPs isolated from cell lysate was undertaken. Methods utilizing three orthogonal and quantitative techniques were developed to suggest that encapsulation of non-viral molecules (nucleic acids or proteins) during in vivo assembly causes distinct changes to the size distribution of isolated VLPs: transmission electron microscopy (TEM), asymmetrical flow field-flow fractionation with multiple-angle light scattering (AFFFF-MALS) and electrospray differential mobility analysis (ES-DMA). The understanding gained from the research presented in this work enables the enhanced capture of partially assembled virus shell protein from cell lysate, as well as a method to efficiently and cost-effectively analyse VLP solutions for the presence of desirable or undesirable encapsulated material.

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation. Includes bibliographical references.

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation. Includes bibliographical references.