Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations
- 12 Downloads
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 functional 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 modified by site-directed mutagenesis to reproduce the LDLR missense mutations most common in the population of northwest Russia so as to study the subcellular localization and function of the modified chimeric protein.
Keywordsgene expression green fluorescent protein familial hypercholesterolemia fusion protein gene expression low density lipoprotein receptor
enhanced green fluorescent protein
low density lipoprotein
phosphate buffer solution
Unable to display preview. Download preview PDF.
- Etxebarria, A., Benito-Vicente, A., Alves, A.C., Ostolaza, H., Bourbon, M., and Martin, C., Advantages and versatility of fluorescence-based methodology to characterize the functionality of LDLR and class mutation assignment, PLoS One, 2014, vol. 9, p. e112677.CrossRefPubMedPubMedCentralGoogle Scholar
- Goldstein, J.L., Hobbs, H.H., and Brown, M.S., Familial hypercholesterolaemia, in The Metabolic and Molecular Basis of Inherited Disease, New York: McGraw Hill, 2001, vol. 3, pp. 2863–2914.Google Scholar
- Komarova, T.Yu., Golovina, A.S., Grudinina, N.A., Zakharova, F.M., Korneva, V.A., Lipovetsky, B.M., Serebrenitskaya, M.P., Konstantinov, V.O., Vasilyev, V.B., and Mandelshtam, M.Yu., New mutations in low density lipoprotein receptor gene in familial hypercholesterolemia patients from Petrozavodsk, Russ. J. Genet., 2013, vol. 49, no. 6, pp. 673–676.CrossRefGoogle Scholar
- Korneva, V.A., Kuznetsova, T.Yu., Bogoslovskaya, T.Yu., Polyakov, D.S., Vasilyev, V.B., Orlov, A.V., and Mandelshtam, M.Yu., Cholesterol levels in genetically determined familial hypercholesterolaemia in Russian Karelia, Cholesterol, Article ID 9375818. doi.org/10.1155/2017/9375818Google Scholar
- Solovyov, K.V., Polyakov, D.S., Grudinina, N.A., Egorov, V.V., Morozova, I.V., Aleynikova, T.D., and Shavlovsky, M.M., Expression in E. coli and purification of the fibrillogenic fusion proteins TTR-sfGFP and β2M-sfGFP, Prep. Biochem. Biotechnol., 2011, vol. 41, pp. 337–349.CrossRefPubMedGoogle Scholar
- Voevoda, M.I., Kulikov, I.V., Shakhtshneider, E.V., Maksimov, V.N., Pilipenko, I.V., Tereschenko, I.P., Kobzev, V.F., Romaschenko, A.G., and Nikitin, Yu.P., The spectrum of mutations in the low density lipoprotein receptor gene in the Russian population, Russ. J. Genet., 2008, vol. 44, no. 10, pp. 1191–1194.CrossRefGoogle Scholar
- Zakharova, F.M., Tatishcheva, Yu.A., Golubkov, V.I., Lipovetsky, B.M., Konstantinov, V.O., Denisenko, A.D., Faergeman, O., Vasilyev, V.B., and Mandelshtam, M.Yu., Familial hypercholesterolemia in St. Petersburg: diversity of mutations argues against a strong founder effect, Russ. J. Genet., 2007, vol. 43, no. 9, pp. 1046–1052.CrossRefGoogle Scholar