Induced heterodimerization and purification of two target proteins by a synthetic coiled-coil tag.
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Induced heterodimerization and purification of two target proteins by a synthetic coiled-coil tag. / Fernandez-Rodriguez, Jesus; Marlovits, Thomas.
In: PROTEIN SCI, Vol. 21, No. 4, 4, 2012, p. 511-519.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Induced heterodimerization and purification of two target proteins by a synthetic coiled-coil tag.
AU - Fernandez-Rodriguez, Jesus
AU - Marlovits, Thomas
PY - 2012
Y1 - 2012
N2 - A synthetic de novo designed heterodimeric coiled-coil was used to copurify two target fluorescent proteins, Venus and enhanced cyan fluorescent protein (ECFP). The coiled-coil consists of two 21-amino acid repetitive sequences, (EIAALEK)(3) and (KIAALKE)(3), named E3 and K3, respectively. These sequences were fused to the C-termini of ECFP or Venus followed by either a strep- or a his-tag, respectively, for affinity purification. Mixed lysates of Venus-K3 and ECFP-E3 were subjected to consecutive affinity purification and showed highly specific association between the coiled-coil pair by SDS-PAGE, gel filtration, isothermal titration calorimetry (ITC), and fluorescence resonance energy transfer (FRET). The tagged proteins eluted as heterodimers at the concentrations tested. FRET analysis further showed that the coiled-coil pair was stable in buffers commonly used for protein purification, including those containing high salt concentration and detergent. This study shows that the E3/K3 pair is very well suited for the copurification of two target proteins expressed in vivo because of its high specificity: it forms exclusively heterodimers in solution, it does not interact with any cellular proteins and it is stable under different buffer conditions.
AB - A synthetic de novo designed heterodimeric coiled-coil was used to copurify two target fluorescent proteins, Venus and enhanced cyan fluorescent protein (ECFP). The coiled-coil consists of two 21-amino acid repetitive sequences, (EIAALEK)(3) and (KIAALKE)(3), named E3 and K3, respectively. These sequences were fused to the C-termini of ECFP or Venus followed by either a strep- or a his-tag, respectively, for affinity purification. Mixed lysates of Venus-K3 and ECFP-E3 were subjected to consecutive affinity purification and showed highly specific association between the coiled-coil pair by SDS-PAGE, gel filtration, isothermal titration calorimetry (ITC), and fluorescence resonance energy transfer (FRET). The tagged proteins eluted as heterodimers at the concentrations tested. FRET analysis further showed that the coiled-coil pair was stable in buffers commonly used for protein purification, including those containing high salt concentration and detergent. This study shows that the E3/K3 pair is very well suited for the copurification of two target proteins expressed in vivo because of its high specificity: it forms exclusively heterodimers in solution, it does not interact with any cellular proteins and it is stable under different buffer conditions.
KW - Amino Acid Sequence
KW - Molecular Sequence Data
KW - Substrate Specificity
KW - Amino Acid Motifs
KW - Electrophoresis, Polyacrylamide Gel
KW - Cloning, Molecular
KW - Chromatography, Affinity
KW - Protein Interaction Mapping
KW - Protein Denaturation
KW - Protein Stability
KW - Fluorescence Resonance Energy Transfer
KW - Buffers
KW - Affinity Labels/chemistry
KW - Calorimetry/methods
KW - Escherichia coli/chemistry
KW - Green Fluorescent Proteins/chemistry/isolation & purification
KW - Plasmids/chemistry
KW - Protein Multimerization
KW - Recombinant Fusion Proteins/chemistry/isolation & purification
KW - Repetitive Sequences, Amino Acid
KW - Salts/chemistry
KW - Solutions/chemistry
KW - Amino Acid Sequence
KW - Molecular Sequence Data
KW - Substrate Specificity
KW - Amino Acid Motifs
KW - Electrophoresis, Polyacrylamide Gel
KW - Cloning, Molecular
KW - Chromatography, Affinity
KW - Protein Interaction Mapping
KW - Protein Denaturation
KW - Protein Stability
KW - Fluorescence Resonance Energy Transfer
KW - Buffers
KW - Affinity Labels/chemistry
KW - Calorimetry/methods
KW - Escherichia coli/chemistry
KW - Green Fluorescent Proteins/chemistry/isolation & purification
KW - Plasmids/chemistry
KW - Protein Multimerization
KW - Recombinant Fusion Proteins/chemistry/isolation & purification
KW - Repetitive Sequences, Amino Acid
KW - Salts/chemistry
KW - Solutions/chemistry
M3 - SCORING: Journal article
VL - 21
SP - 511
EP - 519
JO - PROTEIN SCI
JF - PROTEIN SCI
SN - 0961-8368
IS - 4
M1 - 4
ER -
