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Peptide Tags and Domains for Expression and Detection of Mammalian Membrane Proteins at the Cell Surface

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Heterologous Expression of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2507))

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Abstract

Normal functions of cell-surface proteins are dependent on their proper trafficking from the site of synthesis to the cell surface. Transport proteins mediating solute transfer across the plasma membrane constitute an important group of cell-surface proteins. There are several diseases resulting from mutations in these proteins that interfere with their transport function or trafficking, depending on the impact of the mutations on protein folding and structure. Recent advances in successful treatment of some of these diseases with small molecules which correct the mutations-induced folding and structural changes underline the need for detailed structural and biophysical characterization of membrane proteins. This requires methods to express and purify these proteins using heterologous expression systems. Here, using the solute carrier (SLC) transporter NaCT (Na+-coupled citrate transporter) as an example, we describe experimental strategies for this approach. We chose this example because several mutations in NaCT, distributed throughout the protein, cause a severe neurologic disease known as early infantile epileptic encephalopathy-25 (EIEE-25). NaCT was modified with various peptide tags, including a RGS-His10, a Twin-Strep, the SUMOstar domain, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. When transiently expressed in HEK293 cells, recombinant NaCT proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited citrate transport activity similar to the nontagged protein. Surface NaCT expression was enhanced by the presence of SUMOstar on the N-terminus. The dual-purpose peptide epitopes RGS-His10 and Twin-Strep facilitated detection of NaCT by immunohistochemistry and western blot and may serve useful tags for affinity purification. This approach sets the stage for future analyses of mutant NaCT proteins that may alter protein folding and trafficking. It also demonstrates the capability of a transient mammalian cell expression system to produce human NaCT of sufficient quality and quantity to augment future biophysical and structural studies and drug discovery efforts.

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Acknowledgments

This work was supported by the Cystic Fibrosis Foundation Therapeutics grants URBATS13XX0 to ILU, and National Institute of Health grant R41 AR074854 to VG. We thank the Imaging Core Facilities at TTUHSC for access to the confocal microscope, Dr. Souad Sennoune for proficient help with microscopy and members of the Center for Membrane Protein Research for insightful discussions.

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Authors and Affiliations

  1. Departments of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX, USA

    Valeria Jaramillo-Martinez

  2. Cell Biology and Biochemistry, and the Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA

    Vadivel Ganapathy & Ina L. Urbatsch

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  1. Valeria Jaramillo-Martinez
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  2. Vadivel Ganapathy
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  3. Ina L. Urbatsch
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Correspondence to Ina L. Urbatsch .

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Editors and Affiliations

  1. Université Côte d’Azur, CNRS UMR7275, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France

    Isabelle Mus-Veteau

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Jaramillo-Martinez, V., Ganapathy, V., Urbatsch, I.L. (2022). Peptide Tags and Domains for Expression and Detection of Mammalian Membrane Proteins at the Cell Surface. In: Mus-Veteau, I. (eds) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology, vol 2507. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2368-8_18

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  • DOI: https://doi.org/10.1007/978-1-0716-2368-8_18

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