Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations
- Authors: Polyakov D.S.1, Grudinina N.A.1, Bogoslovskaya T.Y.1, Sokolov A.V.1,2, Mandelshtam M.Y.1,3, Vasilyev V.B.1,2
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Affiliations:
- Institute of Experimental Medicine
- Department of Fundamental Problems of Medicine
- Department of Biochemistry
- Issue: Vol 12, No 2 (2018)
- Pages: 153-159
- Section: Article
- URL: https://journals.rcsi.science/1990-519X/article/view/212625
- DOI: https://doi.org/10.1134/S1990519X18020098
- ID: 212625
Cite item
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 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.
About the authors
D. S. Polyakov
Institute of Experimental Medicine
Email: biochemsokolov@gmail.com
Russian Federation, St. Petersburg, 197376
N. A. Grudinina
Institute of Experimental Medicine
Email: biochemsokolov@gmail.com
Russian Federation, St. Petersburg, 197376
T. Yu. Bogoslovskaya
Institute of Experimental Medicine
Email: biochemsokolov@gmail.com
Russian Federation, St. Petersburg, 197376
A. V. Sokolov
Institute of Experimental Medicine; Department of Fundamental Problems of Medicine
Author for correspondence.
Email: biochemsokolov@gmail.com
Russian Federation, St. Petersburg, 197376; St. Petersburg, 199034
M. Yu. Mandelshtam
Institute of Experimental Medicine; Department of Biochemistry
Email: biochemsokolov@gmail.com
Russian Federation, St. Petersburg, 197376; St. Petersburg, 199034
V. B. Vasilyev
Institute of Experimental Medicine; Department of Fundamental Problems of Medicine
Email: biochemsokolov@gmail.com
Russian Federation, St. Petersburg, 197376; St. Petersburg, 199034