Engineered $\beta$-lactoglobulin produced in E-coli : purification, biophysical and structural characterisation
| Autor: | Aneta Łach, Eryk Ludwin, Agnieszka Polit, J.I. Loch, Piotr Bonarek, Krzysztof Lewiński, Barbara Hawro, Magdalena Tworzydło |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Models Molecular ligand binding Bioengineering Lactoglobulins Protein Engineering β-Lactoglobulin Applied Microbiology and Biotechnology Biochemistry law.invention 03 medical and health sciences FLAG-tag law Protein purification Protein A/G Protein stability Escherichia coli Animals Amino Acid Sequence Binding site Molecular Biology Ligand binding N-terminal methionine Original Paper Binding Sites 030102 biochemistry & molecular biology biology Chemistry Circular Dichroism protein engineering Protein engineering Recombinant Proteins Transport protein 030104 developmental biology protein stability Mutation biology.protein Recombinant DNA Thermodynamics Cattle Protein G Protein Binding Biotechnology |
| Zdroj: | Molecular Biotechnology |
| Popis: | 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 material The online version of this article (doi:10.1007/s12033-016-9960-z) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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