Browsing by Author "Middelberg, APJ"

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    Analysis of monoPEGylated human galectin-2 by small-angle x-ray and neutron scattering: concentration dependence of PEG conformation in the conjugate
    (American Chemical Society, 2010-12-01) He, LH; Wang, H; Garamus, VM; Hanley, TL; Lensch, M; Gabius, HJ; Fee, CJ; Middelberg, APJ
    Protein conjugation with polyethylene glycol (PEG) is a valuable means for improving stability, solubility, and bioavailability of pharmaceutical proteins. Using human galectin-2 (hGal-2) and 5 kDa PEG as a model system we first produced a PEG-hGal-2 conjugate exclusively at the Cys75 residue, resulting in two monosubstituted subunits per hGal-2 homodimer. Small angle X-ray and neutron scattering (SAXS and SANS) were combined to provide complementary structural information about the PEG-hGal-2 conjugate, wherein signal generation in SAXS depends mainly on the protein while SANS data presents signals from both the protein and PEG moieties. SAXS data gave a constant radius of gyration (Rg = 21.5 Å) for the conjugate at different concentrations and provided no evidence for an alteration of homodimeric structure or hGal-2 ellipsoidal shape upon PEGylation. In contrast, SANS data revealed a concentration dependence of Rg for the conjugate, with the value decreasing from 31.5 Å at 2 mg/mL to 26 Å at 14 mg/mL (based on hGal-2 concentration). Scattering data have been successfully described by the model of the ellipsoidal homogeneous core (hGal-2) attached with polymer chains (PEG) at the surface. Evidently, the PEG conformation of the conjugate strongly depends on conjugate concentration and PEG’s radius of gyration decreases from 24.5 to 15 Å. An excluded volume effect, arising from steric clashes between PEG molecules at high concentration, was quantified by estimating the second virial coefficient, A2, of PEGylated hGal-2 from the SANS data. A positive value of A2 (6.0 ± 0.4 × 10−4 cm3 mol g−2) indicates repulsive interactions between molecules, which are expected to protect the PEGylated protein against aggregation. © 2010 American Chemical Society
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    The effects of acid hydrolysis on protein biosurfactant molecular, interfacial, and foam properties: pH responsive protein hydrolysates
    (Royal Society of Chemistry, 2012-03-23) Dimitrijev-Dwyer, M; He, LZ; James, M; Nelson, A; Wang, LG; Middelberg, APJ
    The success of hydrolysis in improving the functional foaming properties of surface-active proteins is usually attributed to three factors: decreased molecular size; increased hydrophobicity; and microchemical changes, specifically deamidation of glutamine and asparagine. Studying these individual factors is difficult using naturally-occurring proteins, as hydrolysate products are complex mixed systems, and the mechanisms of foam stabilization are likewise complex. To address this complexity we report studies of a recombinant protein (DAMP4) which comprises four peptide surfactant (DAMP1) molecules connected by acid-labile amino acid (Asp-Pro) linkers. Hydrolysis of DAMP4 under conditions of low pH and high temperature produced h-DAMP1, a mixture of deamidated variants of the chemically-synthesized DAMP1 peptide surfactant. By examining foaming performance of these molecules, we are able to isolate the effects of molecule size (DAMP1 vs. DAMP4) and deamidation (h-DAMP1 vs. DAMP1). Molecule size had little effect on foaming for the conditions studied. However, deamidation completely changed foaming behaviour, most likely due to alteration of interfacial charge structure (through deamidation of glutamine to glutamic acid) and consequent effects on thin-film stability. Good foaming was observed only at pH values away from the isoelectric points (pI) of the biomolecules where an electrostatic barrier to film rupture can occur. The addition of Zn2+ to DAMP4, h-DAMP1 and DAMP1 caused visible aggregation under all conditions, which assisted in stabilising foams only in situations where a net charge would be expected. © 2012, Royal Society of Chemistry
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    High yield expression and efficient purification of deuterated human protein galectin-2
    (Elsevier, 2012-07-01) Chen, XJ; Wilde, KL; Wang, H; Lake, V; Holden, PJ; Middelberg, APJ; He, LH; Duff, AP
    Structural studies of biological macromolecules often require deuterated proteins, necessitating an effective bioprocessing strategy for high yield deuteration and purification. The fermentation and bioseparation studies reported here concern deuterated human protein galectin-2 mutant C57M (hGal-2), a protein showing potential for therapeutic applications. Using the vector pET-28a and a defined D2O based minimal medium with glycerol as the sole carbon source and kanamycin for selection, we have demonstrated that a high density of Escherichia coli expressing deuterated protein at a bench bioreactor scale (7L) can be achieved, with due attention to prevention of oxygen limitation. Yields achieved were 58 g\L biomass (wet weight) containing 0.7 g/L hGal-2. Affinity chromatography and ion-exchange chromatography were combined to achieve high purity as well as removal of hGal-2 aggregates, giving an overall yield of 1200 mg deuterated hGal-2. The deuterated hGal-2 was characterized and compared with the non-deuterated protein by size exclusion chromatography (SEC), HPLC, N-terminal sequencing, mass spectrometry (MS) and a dot blot immunoassay, showing that deuteration and subsequent purification did not impact the lactose binding and antibody recognition abilities of hGal-2. MS for both intact and trypsin-digested hGal-2 demonstrated that the extent of labeling of non-exchangeable hydrogen atoms by deuterium was (66 +/- 1)%, which provides sufficient contrast variation for structural studies using small angle neutron scattering. The fermentation and bioseparation method established in this work can be applied to process other deuterated proteins with high yield and purity, opening the way to advanced structural studies. © Institution of Chemical Engineers 2014.
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    Insights into the role of protein molecule size and structure on interfacial properties using designed sequences
    (Royal Society of Chemistry, 2013-03-06) Dwyer, MD; He, L; James, M; Nelson, A; Middelberg, APJ
    Mixtures of a large, structured protein with a smaller, unstructured component are inherently complex and hard to characterize at interfaces, leading to difficulties in understanding their interfacial behaviours and, therefore, formulation optimization. Here, we investigated interfacial properties of such a mixed system. Simplicity was achieved using designed sequences in which chemical differences had been eliminated to isolate the effect of molecular size and structure, namely a short unstructured peptide (DAMP1) and its longer structured protein concatamer (DAMP4). Interfacial tension measurements suggested that the size and bulk structuring of the larger molecule led to much slower adsorption kinetics. Neutron reflectometry at equilibrium revealed that both molecules adsorbed as a monolayer to the air–water interface (indicating unfolding of DAMP4 to give a chain of four connected DAMP1 molecules), with a concentration ratio equal to that in the bulk. This suggests the overall free energy of adsorption is equal despite differences in size and bulk structure. At small interfacial extensional strains, only molecule packing influenced the stress response. At larger strains, the effect of size became apparent, with DAMP4 registering a higher stress response and interfacial elasticity. When both components were present at the interface, most stress-dissipating movement was achieved by DAMP1. This work thus provides insights into the role of proteins' molecular size and structure on their interfacial properties, and the designed sequences introduced here can serve as effective tools for interfacial studies of proteins and polymers. © 2013, The Royal Society