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


ABSTRACT: Functional recombinant bovine ?-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 ?-lactoglobulin such as CD spectra, ligand binding (n, K a, ?H, T?S, ?G), chemical and thermal stability (?G D, C mid) 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.

SUBMITTER: Loch JI 

PROVIDER: S-EPMC5035327 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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

Loch Joanna I JI   Bonarek Piotr P   Tworzydło Magdalena M   Polit Agnieszka A   Hawro Barbara B   Łach Aneta A   Ludwin Eryk E   Lewiński Krzysztof K  

Molecular biotechnology 20161001 10


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 enable  ...[more]

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