Expression of bovine β-lactoglobulin as a fusion protein in Escherichia coli: a tool for investigating how structure affects function

Ariyaratne, K.A.N.S.; Brown, Rosemary; Dasgupta, Arjit; Jonge, Jørgen de; Jameson, Geoffrey B.; Loo, Trevor S.; Weinberg, Clarice R.; Norris, Gillian E.

doi:10.1016/s0958-6946(02)00027-4

Cited by 10 publications

(3 citation statements)

References 29 publications

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“…A full description of the protein expression and purification processes (67,68), AUC experiments (36,69), and continuum electrostatic calculations (23,(70)(71)(72)(73)(74)(75)(76) is provided in the Supporting Material, together with the accompanying Fig. S1 S2, Table S3, Table S4, Table S5, Table S6, and Table S7.…”

Section: Methodsmentioning

confidence: 99%

Bovine β-Lactoglobulin Is Dimeric Under Imitative Physiological Conditions: Dissociation Equilibrium and Rate Constants over the pH Range of 2.5–7.5

Mercadante

Melton

Norris

et al. 2012

Biophysical Journal

140

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The oligomerization of β-lactoglobulin (βLg) has been studied extensively, but with somewhat contradictory results. Using analytical ultracentrifugation in both sedimentation equilibrium and sedimentation velocity modes, we studied the oligomerization of βLg variants A and B over a pH range of 2.5-7.5 in 100 mM NaCl at 25°C. For the first time, to our knowledge, we were able to estimate rate constants (k(off)) for βLg dimer dissociation. At pH 2.5 k(off) is low (0.008 and 0.009 s(-1)), but at higher pH (6.5 and 7.5) k(off) is considerably greater (>0.1 s(-1)). We analyzed the sedimentation velocity data using the van Holde-Weischet method, and the results were consistent with a monomer-dimer reversible self-association at pH 2.5, 3.5, 6.5, and 7.5. Dimer dissociation constants K(D)(2-1) fell close to or within the protein concentration range of ∼5 to ∼45 μM, and at ∼45 μM the dimer predominated. No species larger than the dimer could be detected. The K(D)(2-1) increased as |pH-pI| increased, indicating that the hydrophobic effect is the major factor stabilizing the dimer, and suggesting that, especially at low pH, electrostatic repulsion destabilizes the dimer. Therefore, through Poisson-Boltzmann calculations, we determined the electrostatic dimerization energy and the ionic charge distribution as a function of ionic strength at pH above (pH 7.5) and below (pH 2.5) the isoelectric point (pI∼5.3). We propose a mechanism for dimer stabilization whereby the added ionic species screen and neutralize charges in the vicinity of the dimer interface. The electrostatic forces of the ion cloud surrounding βLg play a key role in the thermodynamics and kinetics of dimer association/dissociation.

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Section: Methodsmentioning

confidence: 99%

Bovine β-Lactoglobulin Is Dimeric Under Imitative Physiological Conditions: Dissociation Equilibrium and Rate Constants over the pH Range of 2.5–7.5

Mercadante

Melton

Norris

et al. 2012

Biophysical Journal

140

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“…More recent studies showed that BlgB can be produced and secreted by Lactobacillus casei [ 10 ] while attempts to express β-lactoglobulin in E. coli have continued to fail due to incorrect protein folding. The expression systems used for lactoglobulin production were reviewed in details by Ariyaratne et al [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

Engineered β-Lactoglobulin Produced in E. coli: Purification, Biophysical and Structural Characterisation

et al. 2016

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Functional recombinant bovine β-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained. The use of gel filtration on Sephadex G75 at the last purification step enables protein without endogenous ligand to be obtained. The physicochemical properties of recombinant β-lactoglobulin such as CD spectra, ligand binding (n, Ka, ΔH, TΔS, ΔG), chemical and thermal stability (ΔGD, Cmid) and crystal structure confirmed that the protein obtained is almost identical to the natural one. The substitutions of N-terminal residues did not influence the binding properties of the recombinant protein so that the lactoglobulin produced and purified according to our protocol is a good candidate for further engineering and potential use in pharmacology and medicine.Electronic supplementary materialThe online version of this article (doi:10.1007/s12033-016-9960-z) contains supplementary material, which is available to authorized users.

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“…The generation of BLG lysine mutants was the next logical step. However, the overexpression of BLG in Escherichia coli posed a bit of a challenge; despite its long history of investigation, a survey of the recombinant BLG literature revealed significant difficulty expressing soluble and properly folded protein in this common bacterial host. − For our purposes, we had to overcome an additional barrier of low-yielding cultures, as ring-fused dimer formation is slow and requires gram quantities of protein to generate sufficient product for analysis. While soluble BLG expression is successful in yeast systems like Pichia pastoris , cloning, transformation, and expression are much more time-consuming than in E. coli , rendering P. pastoris suboptimal for generating numerous lysine mutants.…”

mentioning

confidence: 99%

Lipofuscin Formation Catalyzed by the Milk Protein β-Lactoglobulin: Lysine Residues in Cycloretinal Synthesis

et al. 2017

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Lipofuscins are toxic autofluorescent byproducts of the visual cycle. The accumulation of lipofuscins such as cycloretinal in the retina is thought to play a role in the progression of age-related macular degeneration (AMD). Intriguingly, the milk protein β-lactoglobulin (BLG) can promote the cyclodimerization of all-trans-retinal to cycloretinal both in vitro and in vivo. Here, site-directed mutagenesis of BLG and mass spectrometric analysis with substrate analogues demonstrate that lysine residues play a key role in catalysis. It is also shown that catalytic activity necessitates the presence of a physical binding site and cannot be mediated by a peptide chain. These studies provide insight into the mechanism of the cyclodimerization process and provide a model system for biocatalysis and biosynthesis of cycloretinal in vivo. In the long term, these studies may pave the way for drug development and inhibitor design as an early treatment regimen for AMD.

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Expression of bovine β-lactoglobulin as a fusion protein in Escherichia coli: a tool for investigating how structure affects function

Cited by 10 publications

References 29 publications

Bovine β-Lactoglobulin Is Dimeric Under Imitative Physiological Conditions: Dissociation Equilibrium and Rate Constants over the pH Range of 2.5–7.5

Bovine β-Lactoglobulin Is Dimeric Under Imitative Physiological Conditions: Dissociation Equilibrium and Rate Constants over the pH Range of 2.5–7.5

Engineered β-Lactoglobulin Produced in E. coli: Purification, Biophysical and Structural Characterisation

Lipofuscin Formation Catalyzed by the Milk Protein β-Lactoglobulin: Lysine Residues in Cycloretinal Synthesis

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