Code | CSB-YP018198HU |
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The pipeline for the recombinant human Cardiac phospholamban (PLN) production in yeast cells consists of the following consecutive steps: construction of the expression vector encoding the human PLN protein (1-52aa) fused with the N-terminal GST-tag, its transformation into yeast cells, high-density cultivation and induction of the positive cells, cell lysis, and expression analysis. The resulting recombinant human PLN protein is purified from the cell lysate through affinity purification, and its purity is greater than 90% as determined by the SDS-PAGE.
PLN is a crucial protein regulating calcium homeostasis in cardiac muscle cells. It acts as the principal regulator of the Ca2+-ATPase of the cardiac sarcoplasmic reticulum [1]. PLN is an integral membrane protein that modulates cardiac muscle contractility by maintaining calcium balance within cardiomyocytes [2]. It is known to control the activity of the sarcoplasmic reticulum Ca2+ pump, which is essential for calcium-mediated muscle relaxation [3].
Structurally, phospholamban is a 52-amino acid membrane protein that can assemble into a pentamer within the sarcoplasmic reticulum membranes [4]. It contains an α-helical transmembrane segment and a cytoplasmic domain that exhibits structural disorder [5]. Phospholamban is phosphorylated in response to β-adrenergic stimulation, which affects its inhibitory effect on the Ca2+ ATPase [6]. The phosphorylation of phospholamban is important in regulating cardiac contraction and relaxation [7].
Phospholamban also modulates cardiac function. Studies have shown that phospholamban knockout mice exhibit altered intracellular calcium transients and myocardial contractility [8]. Furthermore, phospholamban regulates the cardiac Ca-ATPase (SERCA2) as an inhibitory cofactor [9].
References:
[1] H. Simmerman and L. Jones, Phospholamban: protein structure, mechanism of action, and role in cardiac function, Physiological Reviews, vol. 78, no. 4, p. 921-947, 1998. https://doi.org/10.1152/physrev.1998.78.4.921
[2] J. Zamoon, A. Mascioni, D. Thomas, & G. Veglia, Nmr solution structure and topological orientation of monomeric phospholamban in dodecylphosphocholine micelles, Biophysical Journal, vol. 85, no. 4, p. 2589-2598, 2003. https://doi.org/10.1016/s0006-3495(03)74681-5
[3] R. Brusa, F. Magri, N. Bresolin, G. Comi, & S. Corti, Noncoding rnas in duchenne and becker muscular dystrophies: role in pathogenesis and future prognostic and therapeutic perspectives, Cellular and Molecular Life Sciences, vol. 77, no. 21, p. 4299-4313, 2020. https://doi.org/10.1007/s00018-020-03537-4
[4] I. Arkin, P. Adams, A. Brunger, S. Smith, & D. Engelman, Structural perspectives of phospholamban, a helical transmembrane pentamer, Annual Review of Biophysics and Biomolecular Structure, vol. 26, no. 1, p. 157-179, 1997. https://doi.org/10.1146/annurev.biophys.26.1.157
[5] O. Andronesi, S. Becker, K. Seidel, H. Heise, H. Young, & M. Baldus, Determination of membrane protein structure and dynamics by magic-angle-spinning solid-state nmr spectroscopy, Journal of the American Chemical Society, vol. 127, no. 37, p. 12965-12974, 2005. https://doi.org/10.1021/ja0530164
[6] I. Arkin, M. Rothman, C. Ludlam, S. Aimoto, D. Engelman, K. Rothschildet al., Structural model of the phospholamban ion channel complex in phospholipid membranes, Journal of Molecular Biology, vol. 248, no. 4, p. 824-834, 1995. https://doi.org/10.1006/jmbi.1995.0263
[7] C. Mundiña-Weilenmann, L. Vittone, M. Ortale, G. Cingolani, & A. Mattiazzi, Immunodetection of phosphorylation sites gives new insights into the mechanisms underlying phospholamban phosphorylation in the intact heart, Journal of Biological Chemistry, vol. 271, no. 52, p. 33561-33567, 1996. https://doi.org/10.1074/jbc.271.52.33561
[8] J. Autry and L. Jones, Functional co-expression of the canine cardiac ca2+pump and phospholamban in spodoptera frugiperda (sf21) cells reveals new insights on atpase regulation, Journal of Biological Chemistry, vol. 272, no. 25, p. 15872-15880, 1997. https://doi.org/10.1074/jbc.272.25.15872
[9] M. Tada, M. Yabuki, & T. Toyofuku, Molecular regulation of phospholamban function and gene expressiona, Annals of the New York Academy of Sciences, vol. 853, no. 1, p. 116-129, 1998. https://doi.org/10.1111/j.1749-6632.1998.tb08261.x
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I ask if there is a cleavage site between the GST tag and the protein for item CSB-YP018198HU Recombinant Homo sapiens Cardiac phospholamban.
MEKVQYLTRSAIRRASTIEMPQQARQKLQNLFINFCLILICLLLICIIVMLL
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFRT
I would like to know if the PLN is at the N-terminal of the GST or the GST is at the N terminal of the PLN?
The GST tag is located at the N-terminus of PLN.