Paul A Dalby

University College London, Londinium, England, United Kingdom

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Publications (58)164.68 Total impact

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    ABSTRACT: Human IgG4 antibody shows therapeutically-useful properties compared to the IgG1, IgG2 and IgG3 subclasses. Thus IgG4 does not activate complement, and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we have studied the solution structure of wild-type IgG4(Ser222) and a hinge mutant IgG4(Pro222) in different buffers and temperatures, where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s020,w of 6.6-6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the X-ray radius of gyration RG was unchanged with concentration in 50-250 mM NaCl buffers, while the neutron RG values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves P(r) revealed two peaks, M1 and M2 that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures, in addition to IgG4 dimer formation at high concentration in heavy water. Constrained X-ray and neutron scattering modelling revealed asymmetric solution structures for IgG4(Ser222) with extended hinge structures. The IgG4(Pro222) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement, and clarifies aspects of its stability and function for therapeutic applications.
    The Journal of biological chemistry. 05/2014;
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    ABSTRACT: Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially relevant rates of uptake of C7-C16 n-alkanes. Without alkL expression, native E.coli n-alkane uptake was the rate-limiting step in both the whole-cell bioconversion of C7-C16 n-alkanes and in the activation of a whole-cell alkane biosensor by C10 and C11 alkanes. By coexpression of alkL as a transporter plug-in, specific yields improved by up to 100-fold for bioxidation of >C12 alkanes to fatty alcohols and acids. The alkL protein was shown to be toxic to the host when overexpressed but when expressed from a vector capable of controlled induction, yields of alkane oxidation were improved a further 10-fold (8 g/L and 1.7 g/g of total oxidized products). Further testing of activity on n-octane with the controlled expression vector revealed the highest reported rates of 120 μmol/min/g and 1 g/L/h total oxidized products. This is the first time AlkL has been shown to directly facilitate enhanced uptake of C10-C16 alkanes and represents the highest reported gain in product yields resulting from its use.
    Scientific reports. 01/2014; 4:5844.
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    ABSTRACT: Proteins are the most vital biological functional units in every living cell. Measurement of protein stability is central to understanding their structure, function and role in diseases. While proteins are also sought as therapeutic agents, they can cause diseases by misfolding and aggregation in vivo. Here we demonstrate a novel method to measure protein stability and denaturation kinetics, on unprecedented timescales, through optically-induced heating of nanolitre samples in microfluidic capillaries. We obtain protein denaturation kinetics as a function of temperature, and accurate thermodynamic stability data, from a snapshot experiment on a single sample. We also report the first experimental characterization of optical heating in controlled microcapillary flow, verified by computational fluid dynamics modelling. Our results demonstrate that we now have the engineering science in hand to design integrated all-optical microfluidic chips for a diverse range of applications including in-vitro DNA amplification, healthcare diagnostics, and flow chemistry.
    Scientific Reports 07/2013; 3:2130. · 5.08 Impact Factor
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    ABSTRACT: Solution structures for antibodies are critical to understand function and therapeutic applications. The stability of the solution structure of rabbit IgG in different buffers and temperatures was determined by analytical ultracentrifugationand X-ray and neutron scattering. Rabbit IgG showed a principally monomeric species which is well resolved from small amounts of a dimeric species. The proportion of dimer increased with increased concentration, decreased temperatureand heavy water from 8% to 25% in all buffers except for high salt (250 mM NaCl). The Guinier X-ray radius of gyration R(G)likewise increased with concentration in 137 mM NaCl buffer, but was unchanged in 250 mM NaCl buffer.The Guinier neutron R(G)valuesincreased as the temperature decreased. The X-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2whose positions did not change with concentration to indicate unchanged structures in all these conditions. The maximum dimension increased with concentration because of dimer formation. Constrained scattering modelling reproducibly revealed very similar asymmetric solution structures for monomeric rabbit IgG in different buffers, in which the Fab-Fc and Fab-Fab pairswere separated by maximally-extended hinge structures. The dimer was best modelled by two pairs of Fab regions forming tip-to-tip contacts. The intact rabbit IgG structuresexplained the ability of its two ligands, the Fc receptor and complement C1q, to bind to the top of its Fc region which is fully accessible and unhindered by the Fab regions.
    Journal of Molecular Biology 11/2012; · 3.91 Impact Factor
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    ABSTRACT: The uses of 3-formylbenzoic acid and 4-formylbenzoic acid as molecular probes along with previous and new transketolase mutants revealed the factors governing the rate of reaction between transketolase and aromatic aldehydes. The novel α,α-dihydroxyketones were produced at 15 to 30-fold higher yields and up to 250-fold higher specific activities with D469T TK when compared to those obtained for benzaldehyde.
    Organic & Biomolecular Chemistry 10/2012; · 3.57 Impact Factor
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    ABSTRACT: This paper presents a mixed integer linear programming (MILP) model for the optimal synthesis of chromatographic protein purification processes including the time line in which our target protein product is collected. The model is linearised using piecewise linear approximation strategies and tested on three example protein mixtures, containing up to 13 contaminants and selecting from a set of up to 21 candidate steps. The results are also compared with previous literature models attempting to solve the same problem and show that the proposed approach offers significant gains in computational efficiency without compromising the quality of the solution.
    Chemical Engineering Research and Design 09/2012; 90(9):1262–1270. · 1.93 Impact Factor
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    ABSTRACT: The lyophilization of proteins in microplates, to assess and optimise formulations rapidly, has been applied for the first time to a therapeutic protein and, in particular, one that requires a cell-based biological assay, in order to demonstrate the broader usefulness of the approach. Factorial design of experiment methods were combined with lyophilization in microplates to identify optimum formulations that stabilised granulocyte colony-stimulating factor during freeze drying. An initial screen rapidly identified key excipients and potential interactions, which was then followed by a central composite face designed optimisation experiment. Human serum albumin and Tween 20 had significant effects on maintaining protein stability. As previously, the optimum formulation was then freeze-dried in stoppered vials to verify that the microscale data is relevant to pilot scales. However, to validate the approach further, the selected formulation was also assessed for solid-state shelf-life through the use of accelerated stability studies. This approach allows for a high-throughput assessment of excipient options early on in product development, while also reducing costs in terms of time and quantity of materials required.
    Biotechnology Letters 12/2011; 34(4):641-8. · 1.85 Impact Factor
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    ABSTRACT: The refolding of protein derived from inclusion bodies is often characterized by low yields of active protein. The optimization of the refolding step is achieved empirically and consequently is time-consuming slowing process development. An automated robotic platform has been used to develop a dilution refold process-screening platform upon which a hierarchical set of assays rapidly determine optimal refolding conditions at the microscale. This hierarchy allows the simplest, cheapest, and most generic high-throughput assays to first screen for a smaller subset of potentially high-yielding conditions to take forward for analysis by slower, more expensive, or protein specific assays, thus saving resources whilst maximizing information output. An absorbance assay was used to initially screen out aggregating conditions, followed by an intrinsic fluorescence assay of the soluble protein to identify the presence of native-like tertiary structure, which was then confirmed by an activity assay. Results show that fluorescence can be used in conjunction with absorbance to eliminate low-yielding conditions, leaving a significantly reduced set of conditions from which the highest yielding ones can then be identified with slower and often more costly activity or RP-HPLC assays, thus reducing bottlenecks in high-throughput analysis. The microwell-based automated process sequence with generic hierarchical assays was also used to study and minimize the effect on redox potential or misfolding, of oxygenation due to agitation, before demonstrating that the platform can be used to rapidly collect data and evaluate different refolding conditions to speed up the acquisition of process development data in a resource efficient manner.
    Biotechnology Progress 12/2011; 28(2):435-44. · 1.85 Impact Factor
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    ABSTRACT: We have previously used targeted active-site saturation mutagenesis to identify a number of transketolase single mutants that improved activity towards either glycolaldehyde (GA), or the non-natural substrate propionaldehyde (PA). Here, all attempts to recombine the singles into double mutants led to unexpected losses of specific activity towards both substrates. A typical trade-off occurred between soluble expression levels and specific activity for all single mutants, but many double mutants decreased both properties more severely suggesting a critical loss of protein stability or native folding. Statistical coupling analysis (SCA) of a large multiple sequence alignment revealed a network of nine co-evolved residues that affected all but one double mutant. Such networks maintain important functional properties such as activity, specificity, folding, stability, and solubility and may be rapidly disrupted by introducing one or more non-naturally occurring mutations. To identify variants of this network that would accept and improve upon our best D469 mutants for activity towards PA, we created a library of random single, double and triple mutants across seven of the co-evolved residues, combining our D469 variants with only naturally occurring mutations at the remaining sites. A triple mutant cluster at D469, E498 and R520 was found to behave synergistically for the specific activity towards PA. Protein expression was severely reduced by E498D and improved by R520Q, yet variants containing both mutations led to improved specific activity and enzyme expression, but with loss of solubility and the formation of inclusion bodies. D469S and R520Q combined synergistically to improve k(cat) 20-fold for PA, more than for any previous transketolase mutant. R520Q also doubled the specific activity of the previously identified D469T to create our most active transketolase mutant to date. Our results show that recombining active-site mutants obtained by saturation mutagenesis can rapidly destabilise critical networks of co-evolved residues, whereas beneficial single mutants can be retained and improved upon by randomly recombining them with natural variants at other positions in the network.
    Journal of Biotechnology 11/2011; 157(1):237-45. · 3.18 Impact Factor
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    ABSTRACT: Abstract This work describes an experimental ‘toolbox’ for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental ‘toolbox’, one-pot syntheses of the diastereoisomers (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee( enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this ‘toolbox’ provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.
    09/2011; 29(5):192-203.
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    ABSTRACT: Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution strategies to identify improved variants. Mutants P237S and F251Y were 2.4-fold more active towards N-benzoyl valine relative to the wild type at 65°C. F251 mutations to basic residues resulted in 4.5-11-fold shifts in the substrate preference towards N-benzoyl phenylalanine relative to N-benzoyl valine. The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of k(cat) values for N-benzoyl valine and N-benzoyl phenylalanine. Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance, whereas those improving activity towards N-benzoyl-valine occurred in the hinge region loops linking the dimerization and zinc-binding domains in each monomer. These observations support a previously proposed substrate induced conformational transition between open and closed forms of aminoacylases.
    Journal of Biotechnology 07/2011; 155(4):396-405. · 3.18 Impact Factor
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    ABSTRACT: We have previously shown that the denaturation of TK with urea follows a non-aggregating though irreversible denaturation pathway in which the cofactor binding appears to become altered but without dissociating, then followed at higher urea by partial denaturation of the homodimer prior to any further unfolding or dissociation of the two monomers. Urea is not typically present during biocatalysis, whereas access to TK enzymes that retain activity at increased temperature and extreme pH would be useful for operation under conditions that increase substrate and product stability or solubility. To provide further insight into the underlying causes of its deactivation in process conditions, we have characterised the effects of temperature and pH on the structure, stability, aggregation and activity of Escherichia coli transketolase. The activity of TK was initially found to progressively improve after pre-incubation at increasing temperatures. Loss of activity at higher temperature and low pH resulted primarily from protein denaturation and subsequent irreversible aggregation. By contrast, high pH resulted in the formation of a native-like state that was only partially inactive. The apo-TK enzyme structure content also increased at pH 9 to converge on that of the holo-TK. While cofactor dissociation was previously proposed for high pH deactivation, the observed structural changes in apo-TK but not holo-TK indicate a more complex mechanism.
    Journal of Biotechnology 06/2011; 155(2):209-16. · 3.18 Impact Factor
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    Paul A Dalby
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    ABSTRACT: Directed evolution is widely used to improve enzymes, particularly for industrial biocatalytic processes. Molecular biology advances present many new strategies for directed evolution. Commonly used techniques have led to many successful examples of enzyme improvement, yet there is still a need to improve both the efficiency and capability of directed evolution. Recent strategies aimed at making directed evolution faster and more efficient take better advantage of available structural and sequence information. The underlying principles that lead to early dead-ends for directed evolution experiments are also discussed along with recent strategies designed to by-pass them. Several emerging methods for creating novel enzymes are also discussed including examples of catalytic activity for which there is no precedent in nature. Finally, the combined use of several strategies is likely to be required in practice to improve multiple target properties of an enzyme, as successfully shown by a recent industrial example.
    Current Opinion in Structural Biology 06/2011; 21(4):473-80. · 8.74 Impact Factor
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    ABSTRACT: Chemical denaturation of ligand-protein complexes can provide the basis of a label-free binding assay. Here, we show how the technique can be used as a sensitive/affordable screen of potential ligands from a pool of lead drug variants. To demonstrate, we characterized the binding of polyketide ligands based on the mTOR inhibitor rapamycin to the cellular immunophilin FKBP12. This used the intrinsic fluorescence of the protein to monitor the chemical denaturation of each FKBP12-ligand complex. The assay was then successfully modified to a 96-well plate-based screen. Both formats were able to differentiate binding affinities across a wide dynamic range.
    Analytical Biochemistry 04/2011; 411(1):155-7. · 2.58 Impact Factor
  • Shahina S Ahmad, Paul A Dalby
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    ABSTRACT: Reversible precipitation can be used as an efficient purification tool for proteins. In addition, identifying conditions under which precipitation or aggregation occurs is of key importance in the bioprocessing and pharmaceutical industry, as this can aid in better formulations and hinder aggregation in chromatography. We have evaluated the precipitation of proteins as determined by light scattering in microplates as a tool for the high-throughput determination of thermodynamic parameters for protein precipitation, with the potential for screening of formulation additives and relevant bioprocess conditions such as pH. This provides a useful complementary technique to existing microplate-based protein thermostability measurements. Using hen egg-white lysozyme and alcohol dehydrogenase as model proteins we have determined the extent of reversible precipitation as a function of ammonium sulfate and sodium chloride concentrations, and also demonstrated global fitting of the data to generate a model where the fraction precipitated can be predicted for any given condition. The global fit provided thermodynamic parameters, including the free energy for protein precipitation, and also allowed an approximate determination of the average size of the structural nucleus that contributes to the free energy of precipitation for each protein. The rapid collection of thermodynamic parameters for protein precipitation, in parallel with protein thermostability measurements, will provide a powerful platform for protein formulation, and also lead to datasets useful for testing theoretical predictions of reversible precipitation based on the molecular modeling of specific protein structure interactions.
    Biotechnology and Bioengineering 02/2011; 108(2):322-32. · 4.16 Impact Factor
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    ABSTRACT: Downstream bioprocessing and especially chromatographic steps, commonly used for the purification of multicomponent systems, are significant cost drivers in the production of therapeutic proteins. There has been an increased interest in the development of systematic methods for the design of such processes, and the appropriate selection of a series of chromatographic steps is still a major challenge to be addressed. Several models have been developed previously but have assumed that 100% recovery of the desired product is obtained at each chromatographic step. In this work, a mathematical framework is proposed, based on mixed integer optimisation techniques, that removes this assumption and allows full flexibility on the position of retention time cut-points, between which the desired product fraction is collected. The proposed model is demonstrated on three example protein mixtures, each containing up to 13 contaminants and selecting from a set of up to 21 candidate steps. The proposed model results in a reduction of one to three chromatographic steps over solutions that no losses are allowed.
    Biotechnology Progress 01/2011; 27(6):1653-60. · 1.85 Impact Factor
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    ABSTRACT: Of the four human IgG antibody subclasses IgG1-IgG4, IgG4 is of interest in that it does not activate complement and exhibits atypical self-association, including the formation of bispecific antibodies. The solution structures of antibodies are critical to understand function and therapeutic applications. Thus IgG4 was studied by synchrotron X-ray scattering. The Guinier X-ray radius of gyration R(G) increased from 5.0 nm to 5.1 nm with an increase of concentration. The distance distribution function P(r) revealed a single peak at 0.3 mg/ml, which resolved into two peaks that shifted to smaller r values at 1.3 mg/ml, even though the maximum dimension of IgG4 was unchanged at 17 nm. This indicated a small concentration dependence of the IgG4 solution structure. By analytical ultracentrifugation, no concentration dependence in the sedimentation coefficient of 6.4 S was observed. Constrained scattering modelling resulted in solution structural determinations that showed that IgG4 has an asymmetric solution structure in which one Fab-Fc pair is closer together than the other pair, and the accessibility of one side of the Fc region is masked by the Fab regions. The averaged distances between the two Fab-Fc pairs change by 1-2 nm with the change in IgG4 concentration. The averaged conformation of the Fab regions appear able to hinder complement C1q binding to the Fc region and the self-association of IgG4 through the Fc region. The present results clarify IgG4 function and provide a starting point to investigate antibody stability.
    Biochemical Journal 11/2010; 432(1):101-11. · 4.65 Impact Factor
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    ABSTRACT: Transketolase mutants have been identified that accept aromatic acceptors with good stereoselectivities, in particular benzaldehyde for which the wild type enzyme showed no activity.
    Chemical Communications 10/2010; 46(40):7608-10. · 6.38 Impact Factor
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    ABSTRACT: There is increasing interest in the use of biocatalysts in synthetic applications due to their ability to achieve highly stereoselective and atom efficient conversions. Recent developments in molecular biology techniques and synthetic biology have also enhanced potential applications using non-natural substrates. Here we review strategies to α, α '-dihydroxy ketones and 2-amino-1,3-diols via the use of transketolase, a carbon-carbon bond forming biocatalyst, and the enzyme transaminase which converts aldehydes and ketones to amines. Using an integrated strategy we have investigated new chemistries and assays, identified novel biocatalysts and used directed evolution strategies, together with miniaturization studies and modelling to achieve rapid and predictive process scale-up.
    Current Organic Chemistry 09/2010; 14(17):1883-1893. · 3.04 Impact Factor
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    ABSTRACT: Protein stability and ligand-binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5-20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000-fold to just 10(8) molecules. The stability of wild-type FKBP-12 and a destabilizing binding-pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP-12 and rapamycin originates from residue Phe99 in the binding site.
    Protein Science 08/2010; 19(8):1544-54. · 2.74 Impact Factor