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

Abstract

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.

New ligand-binding sites identified in the crystal structures of β-lactoglobulin complexes with desipramine

“…The CD spectra of FAF and FAW (Supplementary Fig. S2) were compared with the spectrum of the WT recombinant protein, which is identical to that of the natural (native) protein (Loch et al, 2016). The FAF spectrum is consistent with the spectra recorded for other lactoglobulin mutants (Loch et al, 2018), while for FAW a significant increase of the signal is observed in the entire spectrum range.…”

“…The presence of mutations was confirmed by DNA sequencing (Genomed S.A., Poland). The genes for both of the new variants, I56F/L39A/M107F (FAF) and I56F/L39A/M107W (FAW), carried the N-terminal L1A/ I2S substitutions (Loch et al, 2016). The FAF and FAW proteins were expressed and purified according to the previously published protocol #2 (Loch et al, 2016).…”

“…The genes for both of the new variants, I56F/L39A/M107F (FAF) and I56F/L39A/M107W (FAW), carried the N-terminal L1A/ I2S substitutions (Loch et al, 2016). The FAF and FAW proteins were expressed and purified according to the previously published protocol #2 (Loch et al, 2016). Native (natural) BLG was purchased from Merck and was purified by size-exclusion chromatography using a HiLoad 26/600 Superdex 75 column (GE Healthcare).…”

Revealing the Dimeric Crystal and Solution Structure of β-Lactoglobulin at pH 4 and Its pH and Salt Dependent Monomer–Dimer Equilibrium

“…α-2 may thus be required for stabilization of the dimeric state. α-3 (residues 113–116 in the GH loop) was found in the present pH 4.0 crystal structure and in the crystal structure determined at pH 6.5, but lacking in all other reported crystal ,− ,, and NMR structures, , and hence is not well conserved (Table S1). α-4 (residues 153–157 in the C-terminal loop) was similar in the present pH 4.0 and crystal structures at pH 5.2 (2AKQ), pH 6.2 (3BLG), and pH 8.2 (2BLG), one residue longer than in structures at pH 2.0 (1CJ5) and 6.5–7.3 (1BSO, 1BSQ, and 1BEB), ,, one residue shorter than in the NMR structure at pH 2.6 (1DV9) (Table S1), and lacking in the crystal structure at pH 8.5.…”

“…Using PyMOL, the secondary structure elements in the present crystal structure were compared with earlier crystal structures covering the pH range 5.2–8.5; 2AKQ (pH 5.2); 3BLG (pH 6.2); 1BEB (pH 6.5); 1BSQ (pH 7.1); 1BSO (pH 7.3); 2Q2M (pH 7.4); 2BLG (pH 8.2); and 5HTE (pH 8.5) as well as with two NMR structures: 1CJ5 (pH 2.0) and 1DV9 (pH 2.6) (Table S1). Helix α-1 (residues 12–15 in the N-terminal loop) is several residues longer in some of these other BLGA structures and not well conserved in the lipocalin family. , α-2 (residues 29–33 in the AB loop) in previously reported structures was formed by residues 29–32 and is together with strand I situated at the dimer interface and lacking in the NMR monomer structures determined at pH 2.0 (1CJ5) and at pH 2.6 (1DV9) .…”