Measurement and Prediction of Protein Phase Behaviour and Protein-Protein Interactions

Cornelius Faber

    Research output: Book/ReportPh.D. thesisResearch

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    Abstract

    The overall aim of this thesis was to evaluate traditional and novel approaches for the characterisation and quantification of the influence of different solution properties such as changes in salt concentration, pH and temperature on protein solubility, crystal growth and the overall protein phase diagram under process relevant conditions. The tools were evaluated with the aim of reducing protein demand whilst operating in the presence of impurities. Concentrates of two recombinant α-amylases of Bacillus halmapalus (BHA) and Bacillus licheniformis (BLA) which had been purified only to technical grade were used in the experimental part of this thesis.

    The development of optimised microbial strains along with advanced fermentation technologies leads to cleaner industrial fermentation broths with high product concentrations, facilitating the economical production of low-value-proteins in large-scale. At the same time, the risk of operating above the solubility limit and consequent uncontrolled formation of amorphous precipitation or crystals is significantly enhanced and can lead to substantial problems during processing, e.g., the loss of product during commonly employed biomass removal steps such as centrifugation or filtration. The design and the operation of downstream processes under conditions close to the solubility limit are particularly challenging and often rely on trial-and-error due to the absence of comprehensive solubility data for process relevant conditions. Thus information on the solution properties of proteins are of far reaching value
    not only for the design and operation of recovery processes but also in initial screening routines for promising new candidate molecules. However, solubility data are available for a limited number of proteins only, since the traditional determination of solubility curves requires a substantial amount of protein and that their crystallisation is possible within a reasonable time frame, constraints which are often not met (Chapter 1).

    In Chapter 2 solubility properties of BHA were extensively studied. Solubility curves were obtained by classical temperature-controlled batch crystallisation processes conducted in Eppendorf tubes at a working volume of 1 mL. The influence of temperature, pH and selected cations and anions from the Hofmeister series on the solubility was quantified. The solubility was found to be almost insensitive to temperature but strongly dependent on pH. The Hofmeister series for anions was followed in the correct order which was also true for monovalent cations, with the exception of lithium which was expected to be the worst precipitant but found to be the best. Measurements of the zeta potential were conducted and have demonstrated that lithium increased the isoelectric point (pI) of BHA which could explain the unexpected solubility behaviour of the enzyme in the presence of this ion.

    In Chapter 3 batch crystallisation processes of BHA started from three different
    supersaturations were further studied. The crystal size, size distributions and the crystal concentration as well as the protein concentration in the supernatant were measured as a function of time. A significant number of crystals already formed during pH-adjustment to induce the supersaturation. Within the first two to three hours of the process, the crystal concentration further increased but then remained constant for a certain period, the length of which was determined by the supersaturation. Only if the supersaturation was high enough, the crystal concentration further increased and reached equilibrium within the experimental
    run time of maximum 48 hours. Contrary to the crystal concentration, the crystal size distribution did not change during the process and the mean diameter of the crystals remained constant and ranged between 4 and 5 µm such that no continuous growth could be observed. The crystal size distribution was essentially independent of the supersaturation.

    The influence of selected cations and anions from the Hofmeister series on the solubility of BLA is presented in Chapter 4. In contrast to BHA, BLA is stable on both sides of the pI, allowing testing of the hypothesis that the Hofmeister series is reversed depending on the sign of the protein net charge. The solubility of BLA was measured at pH 6, 7 and 8 at salt concentrations of 0 M, 0.1 M and 0.5 M. The sign of the protein net charge in the saltsolutions was determined by measuring the zeta potential, at the above mentioned pH-values in the presence of 0 M and 0.1 M salt only. Zeta potential measurements became inaccurate at
    salt concentrations higher than 0.1 M, thus the resulting pI at 0.5 M could not be determined. With the exception of 0.5 M sodium thiocyanate, a minimum in solubility at pH 7 was found for all of the measured conditions. The effect of anions on α-amylase solubility was observed to follow the Hofmeister series. A reversal of the Hofmeister series for cations and anions depending on the sign of the protein net charge could not conclusively be demonstrated.

    The potential of microtitre plates to generate BHA-phase diagrams consisting of precipitation and nucleation zones, as well as metastable and undersaturated zones is demonstrated in Chapter 5. Temperature-controlled micro-batch experiments were conducted at working volumes of 200 µL and the precipitation behaviour analysed by light microscopy. The formation of amorphous precipitate and crystals of different habit was related to the corresponding starting conditions allowing different zones describing the precipitation behaviour to be defined. Inspection of the microtitre plates was conducted after four days and
    the solubility data presented in chapter 2 were incorporated. Phase diagrams were recorded as a function of pH, and of the concentration of sodium chloride at pH 7 and 9 and of sodium thiocyanate at pH 7, respectively. In all cases, a wide nucleation zone could be identified. Amorphous precipitate was formed only at very high initial supersaturations. For sodium chloride at pH 9, no amorphous precipitate was found. At the termination of the experiment, the metastable zone was found to be very narrow for sodium chloride at pH 9 and as a function of pH in the absence of added salts. In contrary, the metastable zone broadened with sodium chloride concentration and even more with sodium thiocyanate concentration, both at pH 7.

    Although the protein demand could be significantly be minimised by the methods presented above, the availability of sufficient protein and the crystallisability may still remain constraints which are hard to overcome, thus other methods were examined in Chapter 6 using BHA as model protein. Determination of the second osmotic virial coefficient (B22) is an attractive alternative to solubility measurements and offers the distinct advantage that experiments can be conducted in undersaturated solutions so that little protein and no timeconsuming crystallisation processes are needed. The B22 represents a Boltzmann-weighted average measure of the protein-protein interactions where positive B22-values correspond to repulsive interactions, while negative values correspond to attractive interactions, thus allowing for a prediction of phase separation and aggregation processes. Dynamic light scattering (DLS) of the BHA preparation used during the experimental work of this thesis demonstrated the presence of significant amounts of soluble enzyme aggregates which was concluded to be why static light scattering (SLS) did not lead to a reliable determination of B22 values of BHA solutions as used in this study. An alternative method to SLS is selfinteraction chromatography (SIC) which has previously been shown to provide B22-values which agree quantitatively with those made by SLS but is at least one order of magnitude more efficient in terms of protein consumption and time needed. The experiments presented in Chapter 6 confirm the insensitivity of SIC towards aggregates. Furthermore, a large set of
    B22-data was generated and good agreement between B22 and solubility measurements was found for BHA. The Hofmeister series was confirmed for both cations and anions and a minimum in B22 as well as in solubility at pH 9 was found for sodium nitrate and sodium thiocyanate between 0.1 M and 0.2 M. Correlations between solubility and B22 have previously been established e.g., by the Haas-Drenth-Wilson model which in this present thesis was found to be in qualitative agreement with the observed trends for BHA.
    Original languageEnglish
    Place of PublicationKgs. Lyngby
    PublisherTechnical University of Denmark
    Number of pages197
    ISBN (Print)87-91494-45-1
    Publication statusPublished - May 2006

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