Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations

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Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations. / Polyakov, D. S.; Grudinina, N. A.; Bogoslovskaya, T. Yu; Sokolov, A. V.; Mandelshtam, M. Yu; Vasilyev, V. B.

в: Cell and Tissue Biology, Том 12, № 2, 2018, стр. 153-159.

Результаты исследований: Научные публикации в периодических изданиях › статья › Рецензирование

BibTeX

@article{037a30909ff84303b54c6cc7455bd568,

title = "Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations",

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.",

keywords = "familial hypercholesterolemia, fusion protein, gene expression, green fluorescent protein, low density lipoprotein receptor",

author = "Polyakov, {D. S.} and Grudinina, {N. A.} and Bogoslovskaya, {T. Yu} and Sokolov, {A. V.} and Mandelshtam, {M. Yu} and Vasilyev, {V. B.}",

note = "Polyakov, D.S., Grudinina, N.A., Bogoslovskaya, T.Y. et al. Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations. Cell Tiss. Biol. 12, 153–159 (2018). https://doi.org/10.1134/S1990519X18020098",

year = "2018",

doi = "10.1134/S1990519X18020098",

language = "English",

volume = "12",

pages = "153--159",

journal = "Cell and Tissue Biology",

issn = "1990-519X",

publisher = "МАИК {"}Наука/Интерпериодика{"}",

number = "2",

}

RIS

TY - JOUR

T1 - Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations

AU - Polyakov, D. S.

AU - Grudinina, N. A.

AU - Bogoslovskaya, T. Yu

AU - Sokolov, A. V.

AU - Mandelshtam, M. Yu

AU - Vasilyev, V. B.

N1 - Polyakov, D.S., Grudinina, N.A., Bogoslovskaya, T.Y. et al. Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations. Cell Tiss. Biol. 12, 153–159 (2018). https://doi.org/10.1134/S1990519X18020098

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - familial hypercholesterolemia

KW - fusion protein

KW - gene expression

KW - green fluorescent protein

KW - low density lipoprotein receptor

UR - http://www.scopus.com/inward/record.url?scp=85045645800&partnerID=8YFLogxK

U2 - 10.1134/S1990519X18020098

DO - 10.1134/S1990519X18020098

M3 - Article

AN - SCOPUS:85045645800

VL - 12

SP - 153

EP - 159

JO - Cell and Tissue Biology

JF - Cell and Tissue Biology

SN - 1990-519X

IS - 2

ER -

ID: 42247378