153 ISSN 1990-519X, Cell and Tissue Biology, 2018, Vol. 12, No. 2, pp. 153–159. © Pleiades Publishing, Ltd., 2018. Original Russian Text © D.S. Polyakov, N.A. Grudinina, T.Yu. Bogoslovskaya, A.V. Sokolov, M.Yu. Mandelshtam, V.B. Vasilyev, 2017, published in Tsitologiya, 2017, Vol. 59, No. 12, pp. 881–887. Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations D. S. Polyakov a , N. A. Grudinina a , T. Yu. Bogoslovskaya a , A. V. Sokolov a, b, *, M. Yu. Mandelshtam a, c , and V. B. Vasilyev a, b a Institute of Experimental Medicine, St. Petersburg, 197376 Russia b Department of Fundamental Problems of Medicine, St. Petersburg State University, St. Petersburg, 199034 Russia c Department of Biochemistry, St. Petersburg State University, St. Petersburg, 199034 Russia *e-mail: biochemsokolov@gmail.com Received July 27, 2017 Abstract—Mutations in the low density lipoprotein receptor gene (LDLR) frequently impair folding and intracellular traffic of the receptor protein, resulting in the development of a monogenic disorder, familial hypercholesterolemia (FH). Identification of novel LDLR mutations requires confirmation of their func- tional importance in distinguishing pathogenic mutations from neutral changes in the aminoacid sequence. To elaborate a system for evaluation of the effect of mutation on the folding and intracellular transport of the LDLR, as well as its ability to bind low density lipoprotein (LDL), we constructed a plasmid containing LDLR cDNA and the gene of enhanced green fluorescent protein (EGFP). Confocal microscopy has shown that, upon transient transfection of HEK293 cells with the plasmid, the recombinant fusion protein LDLR– EGFP is transported onto the cellular membrane and binds labeled LDL. This construct will be further mod- ified by site-directed mutagenesis to reproduce the LDLR missense mutations most common in the popula- tion of northwest Russia so as to study the subcellular localization and function of the modified chimeric pro- tein. Keywords: gene expression, green fluorescent protein, familial hypercholesterolemia, fusion protein, gene expression, low density lipoprotein receptor DOI: 10.1134/S1990519X18020098 INTRODUCTION Mutations in the low density lipoprotein receptor gene cause familial hypercholesterolemia (FH) (OMIM: 143890), a widespread inherited disease (Goldstein et al., 2001). We have characterized the LDLR mutation spectrum in the population of St. Petersburg, Russia (Zakharova et al., 2007), and Petrozavodsk, Russia (Komarova et al., 2013; Korneva et al., 2017). Most mutations revealed in Russia (Meshkov et al., 2004; Voevoda et al., 2012), as well as worldwide, are missense mutations. The discovery of missense mutations in LDLR, especially in the case of novel variants and small family size, does not allow one to show unambiguously the cosegregation of mutations and disease and requires proof that muta- tion has an effect impairing gene expression or protein function. LDLR defects are divided into five classes: block of ligand (LDL) binding by LDLR, impaired LDLR internalization after its binding with a ligand, dis- rupted LDLR recycling after LDL dissociation, impaired LDLR transport from the endoplasmic reticulum after synthesis, and an insufficient amount of LDLR in cells (Hobbs et al., 1990; Goldstein et al., 2001; Etxebarria et al., 2015). Thus, to analyze LDLR functioning, it is necessary to determine its produc- tion, cell localization, and binding with LDL. LDLR binding is assayed with fluorescent- or radioactive- labeled LDL (Silva et al., 2012; Etxebarria et al., 2014). FH is caused by LDLR mutations, as well as muta- tions in genes of apolipoprotein B-100 (the only pro- tein in the composition of LDL), PCSK9 (proprotein convertase), and LDLRAP1 (LDLR adaptor protein). Thereby, to draw correct conclusions concerning LDLR defects, it is inappropriate to investigate its functioning in cells isolated from patients (Soutar and Naoumova, 2007). LDLR with mutations should be expressed in cells with reduced expression of normal Abbreviations: FH—familial hypercholesterolemia, EGFP— enhanced green fluorescent protein, LDL—low density lipopro- tein, LDLR—LDL receptor, PBS—phosphate buffer solution.