Efficient refolding and reconstitution of tissue factor into nanodiscs facilitates structural investigation of a multicomponent system on a lipid bilayer
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Efficient refolding and reconstitution of tissue factor into nanodiscs facilitates structural investigation of a multicomponent system on a lipid bilayer. / Tidemand, Frederik Grønbæk; Østergaard, Henrik; Ploug, Michael; Kragelund, Birthe B.; Arleth, Lise.
In: Biochimica et Biophysica Acta - Biomembranes, Vol. 1862, No. 6, 183214, 2020.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Efficient refolding and reconstitution of tissue factor into nanodiscs facilitates structural investigation of a multicomponent system on a lipid bilayer
AU - Tidemand, Frederik Grønbæk
AU - Østergaard, Henrik
AU - Ploug, Michael
AU - Kragelund, Birthe B.
AU - Arleth, Lise
PY - 2020
Y1 - 2020
N2 - Structural data on membrane proteins in a lipid membrane environment is challenging to obtain but needed to provide information on the, often essential, protein-lipid interplay. A common experimental bottleneck in obtaining such data is providing samples in sufficient amounts and quality required for structural studies. We developed a new production protocol for the single-pass transmembrane protein (SPTMP) tissue factor (TF), exploiting the high expression level in E. coli inclusion bodies and subsequent refolding. This provided more than 5 mg of functional TF per liter bacterial culture. This is substantially more than what was obtained by the classical approaches for expressing TF in the membrane-anchored configuration. We optimized reconstitution into circularized nanodiscs enabling the formation of stable, TF loaded nanodiscs with different lipid compositions and with a limited material waste. The blood coagulation cascade is initiated by the complex formation between TF and Factor VIIa (FVIIa), and we probed this interaction by a functional assay and SPR measurements, which revealed similar activity and binding kinetics as TF produced by other protocols, demonstrating that high-yield production does not compromise TF function. Furthermore, the amounts of sample produced permitted initial small angle X-ray scattering studies providing the first structural information about TF and its binding to FVIIa in a lipid environment. This strategy possibly allows for probing the multicomponent complex TF:FVIIa together with its substrate Factor X on a lipid bilayer, but may also be relevant as a production strategy for other SPTMP for which structural information, in general, is limited.
AB - Structural data on membrane proteins in a lipid membrane environment is challenging to obtain but needed to provide information on the, often essential, protein-lipid interplay. A common experimental bottleneck in obtaining such data is providing samples in sufficient amounts and quality required for structural studies. We developed a new production protocol for the single-pass transmembrane protein (SPTMP) tissue factor (TF), exploiting the high expression level in E. coli inclusion bodies and subsequent refolding. This provided more than 5 mg of functional TF per liter bacterial culture. This is substantially more than what was obtained by the classical approaches for expressing TF in the membrane-anchored configuration. We optimized reconstitution into circularized nanodiscs enabling the formation of stable, TF loaded nanodiscs with different lipid compositions and with a limited material waste. The blood coagulation cascade is initiated by the complex formation between TF and Factor VIIa (FVIIa), and we probed this interaction by a functional assay and SPR measurements, which revealed similar activity and binding kinetics as TF produced by other protocols, demonstrating that high-yield production does not compromise TF function. Furthermore, the amounts of sample produced permitted initial small angle X-ray scattering studies providing the first structural information about TF and its binding to FVIIa in a lipid environment. This strategy possibly allows for probing the multicomponent complex TF:FVIIa together with its substrate Factor X on a lipid bilayer, but may also be relevant as a production strategy for other SPTMP for which structural information, in general, is limited.
KW - Factor VIIa
KW - Membrane protein
KW - Nanodisc
KW - Refolding
KW - SAXS
KW - Tissue factor
U2 - 10.1016/j.bbamem.2020.183214
DO - 10.1016/j.bbamem.2020.183214
M3 - Journal article
C2 - 32081704
AN - SCOPUS:85080141349
VL - 1862
JO - B B A - Biomembranes
JF - B B A - Biomembranes
SN - 0005-2736
IS - 6
M1 - 183214
ER -
ID: 237844688