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

Molecular Biotechnology (2016) 58(10) 605-618
DOI: 10.1007/s12033-016-9960-z
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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.

Figures

  • Fig. 1 Initial step of lactoglobulin purification on Fractogel TMAE (S) (the example of sBlgB). Proteins were eluted using 0–50 % gradient of 2 M NaCl in 50 mM phosphate pH 6.5. sBlgB was eluted in peak A (fractions marked by arrows on SDSPAGE gel)
  • Fig. 3 Purification of sBlgB#2 on Sephadex G75. Fractions B contained almost pure L1A/ I2S-BlgB
  • Table 1 Statistic of data collection and structure refinement
  • Fig. 4 Far-UV (left panel) and near-UV (right panel) CD spectra of BlgB (solid line, from [27]), rBlgB (dash-dot line), sBlgB#1 (dashed line) and sBlgB#2 (dotted line) in 0.05 M phosphate buffer pH 7.5, 20 C. De was normalised by peptide bonds or protein concentration
  • Fig. 5 Calorimetric isotherms of SDS binding to rBlgB (left panel) and sBlgB#2 (right panel). The experiments were performed at 25 C in 50 mM Tris–HCl buffer pH 7.5 and 50 mM phosphate buffer pH 6.5, respectively. The protein and ligand concentrations were 28 and 500 lM for rBlgB and 37 and 100 lM for sBlgB#2. The solid line represents the best fit of experimental data to the one site binding model. A fixed value of enthalpy (-31.07 kJ/mol) was used in case of rBlgB
  • Table 2 Thermodynamic parameters of SDS binding Ligand binding
  • Fig. 6 Electron density map around endogenous ligand bound in b-barrel of a rBlgB (1.2 r) and b sBlgB#1 (1.0 r). c The position of ligand in sBlgB#1 (cyan) and in milk lactoglobulin complex with myristic acid (orange, PDB: 3UEV). d Electron density map (1.0 r) around residues in empty b-barrel in sBlgB#2 (Color figure online)
  • Fig. 7 a Superposition of sBlgB#2 structure (cyan, C2221) and natural lactoglobulin (orange, PDB: 1B8E, C2221). b Superposition of lactoglobulin structures: rBlgB (chain C: magenta), PDB: 1BSY (blue), PDB: 3BLG (grey), PDB: 1B8E (orange), PDB: 1BEB (yellow) (Color figure online)

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APA

Loch, J. I., Bonarek, P., Tworzydło, M., Polit, A., Hawro, B., Łach, A., … Lewiński, K. (2016). Engineered β-Lactoglobulin Produced in E. coli: Purification, Biophysical and Structural Characterisation. Molecular Biotechnology, 58(10), 605–618. https://doi.org/10.1007/s12033-016-9960-z

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