Protein solubility is a problem for many protein chemists including structural biologists and those developing protein pharmaceuticals. Knowledge of how intrinsic factors influence solubility is limited due to the difficulty in obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, as gels or supersaturated solutions often form, making the determination of solubility values impossible for many proteins. Protein precipitants can be used to obtain comparative solubility measurements, and they fall into three broad classes: salts, long-chain polymers, and organic solvents. Our group has used a model protein, RNase Sa, to create 20 variants that differ by the residues at a single surface-exposed position. We have measured the protein solubility of these variants and have generated an amino acid solubility scale, in the context of a protein, measured in ammonium sulfate. Here, we present solubility scales for these variants using PEG-8000 and isopropanol as precipitants. We find that amino acids can be divided into three groups based on their contribution to protein solubility: those that increase protein solubility, those that decrease protein solubility, and those that show little change in protein solubility as compared to our wild-type protein which has a threonine at the variable position. Of the 20 variants used here, the aspartic acid, glutamic acid, and serine variants show the greatest increases in protein solubility. Based on our results, we propose a strategy for increasing protein solubility: substitute exposed hydrophobic, asparagine, glutamine, and threonine residues with aspartic acid, glutamic acid or serine. To test this hypothesis, we utilize this strategy on a low solubility variant of RNase Sa. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained in the two different precipitants closely agree with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone.

The effects of heat or high pressure treatment on the physicochemical and functional properties of pea proteins were evaluated by measuring polypeptide composition, hydrophobicity, solubility, gelation, emulsification, foaming, water-holding capacity and oil-holding capacity. Heat processing (≥ 70 °C) and high pressure treatment (≥ 200 MPa) led to significant increase (from 1.41 to 2.42) in hydrophobicity of native pea proteins. Native gel electrophoresis showed that the content of 11S protein decreased (increased aggregation) as intensity of pressure treatment was increased. In contrast the 7S protein was resistant to pressure-induced protein aggregation. The solubility and emulsifying capacity of pea proteins processed under higher pressure or heat at neutral pH had slight decreases probably due to the formation of aggregates. High pressure treatment of pea proteins led to reductions in the amount of protease required to produce renin-inhibitory peptides.

Protein engineering is a powerful tool to modify proteins to generate novel and desired properties that could be applied in biotechnological, diagnostics and therapeutic areas. In this thesis, both rational design and library based engineering principles have been exploited to develop affinity proteins with desired traits. One study was focused on the use of site-directed mutagenesis to obtain variants of the staphylococcal protein A-derived 58-residue immunoglobulin binding Z domain with improved affinity for mouse IgG1 Fc. Screening of ca. 170 constructed variants revealed one variant with a single F5I amino acid substitution, denoted ZF5I, with a ten-fold higher affinity. The Fc binding ZF5I variant was further investigated for use in affinity-driven site-specific covalent photoconjugation to mIgG1 monoclonal antibodies. Here, nine candidate positions in the domain were investigated for introduction of a UV-activatable maleimide benzophenone (MBP) group via conjugation to an introduced cysteine residue. The best photo-conjugation results were obtained for a variant in which the MBP was introduced at position 32, denoted ZF5I-Q32C-MBP, which could be conjugated at high yields to all nineteen mouse IgG1s tested. The use of a biotinylated Z-based probe for biotinylation via photoconjugation of a monoclonal anti-interferon gamma antibody resulted in a higher antigen binding activity than if a conventional amine directed biotinylation strategy was used. In a second study, the goal was to develop a new homogeneous immunoassay for quick antigen detection, based on split-protein complementation and pairs of antigen recognizing proteins. In one of the formats investigated, separate fragments of a split-beta-lactamase enzyme reporter were genetically linked to ZF5I-Q32C-MBP units which were individually photo-conjugated to two different mAbs recognizing different epitopes on a human interferon gamma model target analyte. Simultaneous binding of the two mAb-enzyme half probes to the analyte resulted in an analyte concentration-dependent enzyme fragment complementation which could be spectrophotometrically detected using a nitrocefin substrate. Using ribosome display technology, Z-domain based binders to mouse IgG1 were selected from an affibody library. One binder denoted Zmab25 was shown capable of selective binding to mouse IgG in a background of bovine IgG, and could be used for species-selective recovery of monoclonal antibodies from complex samples, resembling hybridoma culture supernatants. Epitope mapping experiments showed that that the binding site on mouse IgG was located in the Fab fragment and was overlapping with that of streptococcal protein G. In a final study, phage display technology was used to select affibodies binding to human interleukin 6 (IL-6), for potential use in rheumatoid arthritis (RA) therapy via blocking of the signaling involving the ternary complex between IL-6, the IL-6 receptor α (IL-6R α) and the gp130 co-receptor. Several affibodies were shown to be capable of blocking the interaction between gp130 and preformed complexes of IL-6 and soluble IL-6R α (IL-6/sIL-6R α) in vitro, corresponding to the so-called trans-signaling interaction. One of these affibody variants denoted ZIL-6_13 showed a KD of approx. 500 pM for IL-6 and was genetically fused to different chain ends of the monoclonal anti-TNF antibody adalimumab to build bi-specific “AffiMab” constructs. One construct, ZIL-6_13-HCAda,in which the affibody was fused to the N-terminus of the adalimumab heavy chain had the most optimal properties in different cell assays and was also evaluated in vivo in an acute serum amyloid A (SAA) mouse RA model, involving a dual challenge of animals with both IL-6 and TNF. Compared to adalimumab that could only reduce SAA levels to 50% at the highest dose, the bi-functional AffiMab reduced SAA levels to below the detection level. / <p>QC 20150416</p>

It is well established that H⁺-coupled dipeptide transport in the cultured intestinal cell line, Caco-2 is electrogenic and results in intracellular acidification, via the apically located transport protein, PepTl. The purpose of this study was to initially investigate both the pH dependency and electrogenic nature of H⁺-coupled dipeptide transport, using microspectrofluorometric analysis of intracellular pH and short circuit current (I_SC) measurement of Gly-Sar transport in Caco-2 monolayers. This aimed to characterise both the apical and basolateral transport mechanisms involved in H⁺-coupled Gly-Sar transport under normal physiological conditions and subsequently investigate how these mechanisms are affected by exposure to pathophysiological manipulators. Transepithelial H⁺-coupled Gly-Sar transport was saturable and displayed typical Michaelis-Menton kinetics in Na⁺-free Krebs buffer. In experiments using an isotonic mannitol buffer, apical Gly-Sar induced similar pH dependent transport kinetics. While the characteristics of basolateral Gly-Sar transport were distinct from PepT1 they were also pH dependent and saturable, indicating that protons are responsible for charge carriage during transepithelial dipeptide transport, via the apical transporter, PepT1 and a kinetically distinct pH-dependent basolateral transporter. The heat stable enterotoxin of <i>E. coli</i>, STa inhibited the capacity of Gly-Sar transport in Caco-2 epithelia as well as inhibiting the Na⁺-dependent recovery from Gly-Sar induced acidification. These effects were also observed with the membrane permeable analogue of cGMP, 8-Br-cGMP, indicating that STa-induced inhibition of H⁺-coupled Gly-Sar transport was mediated via elevation of intracellular cGMP levels. The disruption of STa-dependent, but not 8-Br-cGMP-dependent inhibition of Gly-Sar induced I_SC by 21CIAdo, confirmed this proposition. Additionally, the NO donor, SNP, inhibition Gly-Sar-induced I_SC, with characteristics similar to those observed with 8-Br-cGMP. Using protein kinase inhibitors it emerged that 8-Br-cGMP-dependent inhibition of Gly-Sar transport involved both PKG and PKC, while only PKG was implicated in the STa-dependent inhibition of Gly-Sar transport in these cells.

Protein aggregation is a hallmark of a number of neurodegenerative disorders including Alzheimer???s Disease, Huntington???s Disease, and Amyotrophic Lateral Sclerosis. Despite the common occurrence of protein aggregation in disease, the fundamental mechanisms controlling the propensity of a protein to aggregate are not well understood. Over the past decade, one of the most significant advancements in the field of understanding protein aggregation has been the development of several aggregation prediction algorithms. In this study, two separate approaches were used to investigate the detailed molecular mechanisms of protein aggregation. First, a thorough investigation that compared nine protein aggregation prediction techniques was performed. Protein aggregation propensity calculations were performed on wild type and mutant sequences of three diverse proteins including Superoxide Dismutase (SOD), human Acylphosphatase (AcP), and the amyloid beta peptide (A??42). This study presents the first wide-scale comparison of such a large number of prediction algorithms, and additionally provides new information on the ability of the algorithms to successfully predict the experimentally observed aggregation of several mutations of diverse proteins. The algorithms were predominantly developed based on a set of known amyloid-forming proteins and peptides, however, are quite diverse in the way they were designed and the proteins on which they were tested. Interestingly, significant variation was observed when predicting the aggregation propensity of identical sequences by multiple techniques, indicating that the algorithms do not possess a consensus on the primary factors that govern aggregation. Further analyses compared predicted and observed aggregation data for several mutants of the test proteins. The aggregation prediction algorithms predominantly demonstrated poor to moderate correlations with observed aggregation, and the strongest correlations occurred in instances where the test data was used in the development of the algorithms. The general lack of ability of the algorithms to predict the aggregation patterns of more than one test protein suggests that aggregation may be a much more specific process that it is generally attributed to be in that there may be inherently different properties modulating the aggregation mechanisms of different proteins towards varying aggregate structures. The second component of this project was to experimentally examine the role of salt in influencing protein aggregation as a method to elucidate the specific molecular mechanisms controlling protein aggregation pathways. The ALS-causing SOD1 mutation, A4V, in both the oxidized and reduced apo form, was used as a model protein. The role of NaCl and Na2SO4 in mediating protein aggregation was studied using several techniques. While oxidized apo A4V showed very little evidence of aggregation even in the presence of salt, for reduced apo, aggregates readily formed and were promoted by the addition of salt. This finding correlated with the increasing kosmotropic nature of the salt as described by the Hofmeister series. The aggregates formed in the presence of salt contained intermolecular disulphide bonds and demonstrated ANS and ThT binding, indicating aggregates are likely to be largely hydrophobic and possess beta-sheet morphology. Salt promotes protein aggregation in two ways: 1) electrostatic interactions shield protein charges and reduce repulsion between proteins, and 2) specific interactions stabilize various aggregation-prone conformations of the protein. This work is evidence of the important role of salt in influencing protein aggregation and provides a framework for future studies into the complex effects of solution conditions in modulating protein aggregation pathways. Both aspects of this study contribute greatly to furthering the understanding of the molecular mechanisms governing protein aggregation. This is of particular importance to neurodegenerative diseases, where uncovering the factors that modulate the formation of toxic aggregate species is important for disease treatment and prevention. The potential aggregation mechanisms of SOD1, and the contributions it may play in ALS pathogenesis, will be discussed throughout this study.

The effects of heat or high pressure treatment on the physicochemical and functional properties of pea proteins were evaluated by measuring polypeptide composition, hydrophobicity, solubility, gelation, emulsification, foaming, water-holding capacity and oil-holding capacity. Heat processing (≥ 70 °C) and high pressure treatment (≥ 200 MPa) led to significant increase (from 1.41 to 2.42) in hydrophobicity of native pea proteins. Native gel electrophoresis showed that the content of 11S protein decreased (increased aggregation) as intensity of pressure treatment was increased. In contrast the 7S protein was resistant to pressure-induced protein aggregation. The solubility and emulsifying capacity of pea proteins processed under higher pressure or heat at neutral pH had slight decreases probably due to the formation of aggregates. High pressure treatment of pea proteins led to reductions in the amount of protease required to produce renin-inhibitory peptides.

xiii, 107 leaves : ill. ; 28 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1973) from the Dept. of Biochemistry, University of Adelaide

Fig. C-14 in back pocket / xi, 171, xxxix leaves : ill. ; 25 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.) from the Dept. of Microbiology, University of Adelaide, 1973

Thesis (doctoral)--University of Lund.

Thesis (Ph. D.). UM #9809203. / Title from home page (viewed Nov. 17, 2009). Includes bibliographical references.