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.

A major hurdle facing in vitro protein characterization is obtaining soluble protein from targets that tend to aggregate and form insoluble inclusion bodies. Soluble protein is essential for any biophysical data collection and new methods are needed to approach this significant problem. Directed evolution can be used to discover mutations which lead to improved solubility using an appropriate screening method. Green fluorescent protein (GFP) has been shown to be an effective solubility reporter which can be used to screen for soluble protein variants. We have chosen three diverse enzymes as targets for improving protein solubility using this technique: arachidonate 5-lipoxygenase—an enzyme which converts fatty acids into leukotrienes, PhnG—an enzyme belonging to the bacterial carbon-phosphorus lyase pathway, and RebG—a glycosyltransferase. Error-prone PCR and DNA shuffling were used to generate libraries of mutants which were subsequently cloned into a GFP-fusion screening vector. From the evolution of 5LO and RebG, much was learned about the optimization of the protocols involved in this methodology, including valuable information about how to avoid common “false-positive” results in which fluorescent colonies arise while screening but do not represent an improvement of the target. Evolution of these two targets did not result in an improvement of solubility, however truncation strategies may still prove to be effective, and more work needs to be done in this area. Evolution of PhnG successfully produced one variant, named clone B6, which showed both an improvement in expression and folding over wild type PhnG. It was also discovered that GFPuv can act as an effective solubility enhancing fusion tag for PhnG. Prior to the current studies PhnG had not been effectively expressed and purified in E. coli , however purification and refolding of resolubilized inclusion bodies of the clone B6 PhnG-GFP fusion construct was shown to yield enough soluble protein for future crystallographic studies. / Thesis (Master, Chemistry) -- Queen's University, 2009-10-09 12:26:03.353

Protein solubility plays an important role whether for biophysical and structural studies, or for production and delivery of therapeutic proteins. Poor solubility could lead to protein aggregation, which is an undesired physicochemical mechanism at any stage of recombinant proteins production. To date, more than half of all recombinant therapeutic proteins are produced in mammalian cells, mainly due to the high similarity of the final product to human protein structures. However, poor secretion can occur, due to misfolded proteins or aggregates leading to cellular stress and proteolysis. Another widely-used expression system is E. coli, which can offer a cost-efficient alternative. This system has an important limitation, since proteins tends to form insoluble protein aggregates in the cytoplasm upon heterologous overexpression. Several strategies are being implemented to improved soluble expression, ranging from culture conditions to solubility enhancing tags. However, there is no universal approach or technology that solves protein aggregation. In this thesis two recently published hypotheses from our group have been applied. One stated that soluble expression of proteins was inversely correlated with the size of the largest positively-charged patch on the protein surface. The second hypothesis (of protein solubility), arose from the finding that the relative content of lysine and arginine residues separated E. coli proteins by solubility. Both hypotheses arose from a study of an extensive dataset of experimental solubilities determined for cell-free expression of E. coli proteins. In combination with other widely used strategies, such as lowering expression temperature and inducer concentration, decreasing non-charged (hydrophobic) patches and addition of helical capping for increasing stability, a rational understanding for directed alteration of solubility in a variety of recombinant proteins has been explored. This includes three protein models to test: (i) recombinant human erythropoietin (rHuEPO) (one of the top selling therapeutics) (ii) recombinant 6-Phosphofructo-2-Kinase/fructose-2,6-bisphosphatase (rPFKFB3) (a product for which over-expression has been sought for characterisation and insight into possible cancer therapy) and (iii) a set of three selected E. coli proteins containing high ratios of lysines to arginines: thioredoxin-1 (TRX), cold shock-like protein cspB (cspB), and the histidine-containing phosphocarrier protein (HPr). It was found that single or multiple point mutations (changing amino acids from positive to negative charge or vice versa; or lysines to arginines) verified the predicted effect on rHuEPO, rPFKFB3, TRX, cspB, and HPr solubility (experimentally defined as the distribution between soluble and total fractions) for expression in E. coli. In addition, the redesigned set of rHuEPO transiently expressed in HEK 293-EBNA cells, suggesting that positively-charged patch size may also influence protein secretion. Further application of these computational and experimental approaches could provide a valuable tool in the design and engineering of proteins, with enhanced solubility, stability and secretion.

Abstract not available

This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy. The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days. The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus. Keywords: protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein

<p>This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy. </p><p>The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days. </p><p>The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus.</p><p><b>Keywords:</b> protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein</p>

There is a strong link between solubility, and thus crystallisation, and the molecular interactions of proteins in dilute salt solutions. Such molecular interactions are governed by the weak interaction forces (electrostatic, hydration and hydrophobic). Such forces can be quantitatively estimated in terms of a second virial self-coefficient (B22) and a second virial cross-coefficient (B23) for a single and a binary protein system, respectively. Previous studies confirmed the relation between a value of the second virial coefficient and a type of interaction (attractive or repulsive). The aim of this thesis is to correlate the second virial coefficient with the solubility and nucleation for single and binary protein systems. Model proteins used in this work are lysozyme and ovalbumin from egg-white, and α-amylase from Bacillus Licheniformis (BLA). The measurements are performed for sodium chloride and ammonium sulphate solutions in an acidic pH at 20 oC. Interaction chromatography is used in this work to estimate the B22 and B23 values for the model proteins in salt solutions. From the measured values of B22 and B23, the type of interaction is generalised as a function of the salt type, salt concentration, pH and protein type. For the single protein systems, in ammonium sulphate solutions (0.1 - 2.4 M) at pH 4.0 and 7.0, repulsion or no interactions are observed below 0.8 M and, as the salt concentrations are increased attractive self-interactions are observed for the model proteins. However, for the sodium chloride solutions (0.1 - 2.0 M) at pH 4.0 and 7.0, the interaction patterns vary with the salt concentration, the pH and the type of protein studied. A common feature of the self-interaction for all the model proteins is the attractive interactions close to the isoelectric point. For the binary protein systems, three distinct regions are observed in the ammonium sulphate solutions (0.1 - 1.6 M) at pH in the range 4.0 - 7.0. Attractive or no cross-interactions are observed at low salt concentrations (< 0.5 M). At the intermediate salt concentrations (0.5 - 1.0 M), the cross-interactions are constant and near zero. This is followed by a sharp increase in the attractive interactions above 1.0 M ammonium sulphate concentrations. However, for sodium chloride solutions (0.1 - 1.6 M) at pH 4.0 - 7.0, two distinct regions are observed. Attraction or no interactions are observed at low salt concentrations (< 0.5 M) and above 0.5 M concentrations of sodium chloride, negligible cross-interactions are observed between model proteins. For the single protein system, an overall increase in the solubility of three model proteins is observed with an increase in the concentrations of ammonium sulphate and also for sodium chloride solutions except for BLA, where a salting-in behaviour is observed. Linear regression is used on the solubility data to determine the parameters of the Cohn equation (β and Ks) where the values of β vary with solution pH, protein type and salt type. The values of Ks vary with protein type and salt type. However, it is insensitive to the solution pH for lysozyme in ammonium sulphate, ovalbumin in sodium chloride and BLA in ammonium sulphate solutions. For the binary protein system, the presence of ovalbumin had a measurable effect on lysozyme solubility at pH < 5.0 in both salts. In low concentration sodium chloride solutions (< 0.3 M), a decrease in the solubility of lysozyme was observed with the presence of ovalbumin at acidic pH < 5.0. However, in ammonium sulphate solutions, the lysozyme solubility increases with the addition of ovalbumin in the salt concentration range 1.6 - 2.0 M and at pH < 4.0. The primary nucleation threshold values are also determined for lysozyme in sodium chloride and ammonium sulphate solutions. In sodium chloride solutions (0.2 - 1.0 M), the critical supersaturation values increase as the solution pH is raised from 4.0 to 7.0; however in ammonium sulphate solutions (1.0 - 2.0 M), the reverse effect is observed. The critical supersaturation required to nucleate lysozyme in ammonium sulphate solutions is approximately three times higher than in sodium chloride solutions. For the single protein systems, the measured values of solubility and B22 were correlated using published models (RSL and HDW). For each protein-salt combination, a reasonable single correlation between solubility and B22 is possible as the salt concentrations and pH are varied. There are separate correlations for sodium chloride and ammonium sulphate solutions. Based on the correlation curve of solubility and B22, it is proposed that the acidic pH range (4.0 - 5.0) is better for crystallising and precipitating globular proteins from these salt solutions. If the values of solubility and B22 are converted into a non-dimensional quantity, the data derived from the different protein-salt systems collapse onto a single curve for the same salt type. The B22 values are also correlated with the critical supersaturation (ln(c*/S)) for the primary nucleation of lysozyme in salt solutions. The values of the critical supersaturation increase as the values of the second virial coefficient become negative or reduce. The ideal critical supersaturation required to create nuclei of lysozyme in salt solutions is between 0.1 and 1.4. For the binary protein systems, B23 values were related to the slope of the lysozyme and ovalbumin plot at same salt concentration and solution pH. Further work is required for binary protein systems to generalise such correlations as a function of the salt concentration and pH. The correlations derived in this thesis are useful generally to predict the solubility and primary nucleation of globular protein in salt solutions. This work reinforces the importance of the second virial coefficient in predicting the crystallisation of protein in salt solutions.

A cell is considered to be the smallest unit of life. It carries out a variety of biochemical reactions through the activities of proteins and protein enzymes. In order to perform functions, proteins must be in their native folded state together with the correct environmental conditions. A slight change in pH or temperature could cause disruption to the electrostatic interactions within the protein, thus leading to conformational change and the loss of activity. Studies have shown that solubility could be enhanced by increasing the number of charges on the protein surface. And from the studies of extremophiles, we learned that the presence of non-polar aromatic residues could be a key for thermostable proteins. Thus, charges are important to determine the function and adaptation of proteins.Over the decades, large amount of protein sequence and structure information relating to molecular biology has been produced. By employing algorithms, computational and statistical techniques, it is possible to analyse these data to solve biological problems. Often these investigations are based mainly on sequences since their numbers outstrip the number of available structures. However, adding structures would allow us to investigate problems such as the relationship between charges, sequence, structure and functions, which is the aim of this study.In this thesis, the relationships between proteins and function were examined by various electrostatic features derived from charges and also geometric properties from structures. One interesting finding is that the averaged value of pH of maximum stability of proteins within a subcellular location was highly correlated to the pH of that subcellular compartment, which was due to pKas (of histidines), and their locations on the proteins. We also found that the size of the largest non-charged patch on the protein surface correlates with solubility and provides a predictor with a maximum accuracy of 76%. The use of novel charge-based methods shows little improvement in distinguishing between enzymes and non-enzymes. However, the method of using real charges with grid size of 1 angstrom has paved a way into the idea of using charges and dipoles pattern from enzyme active site to distinguish different enzymes. Finally, a web-tool for displaying conserved residues on 3D protein structure is made available to the public for identifying residues that may be of functional importance.

The number of protein containing therapeutic drugs is growing day by day. Lack of proper storage conditions can cause protein degradation or aggregation. The osmotic second virial coefficient, B22, is a thermodynamic parameter, which can predict protein interaction with other proteins and solvent molecules. B22 has been successfully used as predictor of crystallization conditions for a protein in the solution, and in this study an attempt has been made to relate B22 as a predictor of stability of the protein. Static light scattering was used to measure B22 in our studies. B22 and the solubility of three proteins were measured in several excipient solutions. George et al. in 1997 related the osmotic second virial coefficient with the solubility of protein in a solution. In this study we have attempted to relate solubility with B22 and stability of lysozyme, human serum albumin, and ovalbumin in buffer solutions containing various excipients.

No description available.