title: 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, K a, ΔH, TΔS, ΔG), chemical and thermal stability (ΔG 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.
authors.affiliation: Biocrystallography Group, Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Kraków, Poland.
authors.forename: Joanna I
authors.initials: JI
authors.lastname: Loch
authors.affiliation: Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Kraków, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Bonarek
authors.affiliation: Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Kraków, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Tworzydło
authors.affiliation: Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Kraków, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Polit
authors.affiliation: Biocrystallography Group, Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Kraków, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Hawro
authors.affiliation: Biocrystallography Group, Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Kraków, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Łach
authors.affiliation: Biocrystallography Group, Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Kraków, Poland.
authors.forename: Eryk
authors.initials: E
authors.lastname: Ludwin
authors.affiliation: Biocrystallography Group, Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Kraków, Poland. lewinski@chemia.uj.edu.pl.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Lewiński
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D007782:Lactoglobulins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis; D013816:Thermodynamics
chemical_list: D007782:Lactoglobulins; D011994:Recombinant Proteins
keywords: Escherichia coli; Ligand binding; N-terminal methionine; Protein engineering; Protein stability; β-Lactoglobulin