Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins

Liora Ben-Yair; Rita Slaaby; Asael Herman; Yael Cohen; Eva Biener; Nava Moran; Akihiko Yoshimura; Jonathan Whittaker; Pierre De Meyts; Brian Herman; Arieh Gertler

doi:10.1016/S1046-5928(02)00044-X

Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins

Liora Ben-Yair, Rita Slaaby, Asael Herman, Yael Cohen, Eva Biener, Nava Moran, Akihiko Yoshimura, Jonathan Whittaker, Pierre De Meyts, Brian Herman, Arieh Gertler

Biomedical Engineering

Research output: Contribution to journal › Article › peer-review

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Abstract

To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.

Original language	English (US)
Pages (from-to)	456-464
Number of pages	9
Journal	Protein Expression and Purification
Volume	25
Issue number	3
DOIs	https://doi.org/10.1016/S1046-5928(02)00044-X
State	Published - 2002

Bibliographical note

Funding Information:
We thank Dr. Nils Billestrup from the Hagedorn Research Institute (Denmark) for the gift of bacterial plasmid encoding GST–GHR. Part of this work was conducted during the sabbatical leave of one of the authors (A.G.) at the Hagedorn Research Institute. This work was supported by the Binational USA–Israel Science Foundation (Grant No. 2000115) to A.G. and B.H.

Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.

Keywords

Cyan fluorescent protein
Growth hormone receptor
Prolactin receptor
SOCS proteins
Yellow fluorescent protein

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Ben-Yair, L., Slaaby, R., Herman, A., Cohen, Y., Biener, E., Moran, N., Yoshimura, A., Whittaker, J., De Meyts, P., Herman, B., & Gertler, A. (2002). Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins. Protein Expression and Purification, 25(3), 456-464. https://doi.org/10.1016/S1046-5928(02)00044-X

Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins. / Ben-Yair, Liora; Slaaby, Rita; Herman, Asael et al.
In: Protein Expression and Purification, Vol. 25, No. 3, 2002, p. 456-464.

Research output: Contribution to journal › Article › peer-review

Ben-Yair, L, Slaaby, R, Herman, A, Cohen, Y, Biener, E, Moran, N, Yoshimura, A, Whittaker, J, De Meyts, P, Herman, B & Gertler, A 2002, 'Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins', Protein Expression and Purification, vol. 25, no. 3, pp. 456-464. https://doi.org/10.1016/S1046-5928(02)00044-X

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abstract = "To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.",

keywords = "Cyan fluorescent protein, Growth hormone receptor, Prolactin receptor, SOCS proteins, Yellow fluorescent protein",

author = "Liora Ben-Yair and Rita Slaaby and Asael Herman and Yael Cohen and Eva Biener and Nava Moran and Akihiko Yoshimura and Jonathan Whittaker and {De Meyts}, Pierre and Brian Herman and Arieh Gertler",

note = "Funding Information: We thank Dr. Nils Billestrup from the Hagedorn Research Institute (Denmark) for the gift of bacterial plasmid encoding GST–GHR. Part of this work was conducted during the sabbatical leave of one of the authors (A.G.) at the Hagedorn Research Institute. This work was supported by the Binational USA–Israel Science Foundation (Grant No. 2000115) to A.G. and B.H. Copyright: Copyright 2008 Elsevier B.V., All rights reserved.",

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AU - Herman, Asael

AU - Cohen, Yael

AU - Biener, Eva

AU - Moran, Nava

AU - Yoshimura, Akihiko

AU - Whittaker, Jonathan

AU - De Meyts, Pierre

AU - Herman, Brian

AU - Gertler, Arieh

N1 - Funding Information: We thank Dr. Nils Billestrup from the Hagedorn Research Institute (Denmark) for the gift of bacterial plasmid encoding GST–GHR. Part of this work was conducted during the sabbatical leave of one of the authors (A.G.) at the Hagedorn Research Institute. This work was supported by the Binational USA–Israel Science Foundation (Grant No. 2000115) to A.G. and B.H. Copyright: Copyright 2008 Elsevier B.V., All rights reserved.

PY - 2002

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N2 - To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.

AB - To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.

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KW - Growth hormone receptor

KW - Prolactin receptor

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KW - Yellow fluorescent protein

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Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins

Abstract

Bibliographical note

Keywords

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