Abstract
Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure. Here we describe a simple, one step purification process to purify a large amount of sestrin proteins with significant yield. Further study of recombinant human sestrins may further facilitate the understanding of their roles in eukaryotic cells.
Keywords:
CD; SPR; Sestrin; prokaryotic system.
MeSH terms
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Circular Dichroism
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Escherichia coli / genetics
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Genetic Vectors
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HeLa Cells
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Heat-Shock Proteins / biosynthesis*
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Heat-Shock Proteins / chemistry
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Heat-Shock Proteins / genetics
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Humans
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Mechanistic Target of Rapamycin Complex 1
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Multiprotein Complexes / genetics*
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Multiprotein Complexes / metabolism
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Nuclear Proteins / biosynthesis*
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Protein Conformation, alpha-Helical
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Reactive Oxygen Species / metabolism*
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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TOR Serine-Threonine Kinases / genetics*
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TOR Serine-Threonine Kinases / metabolism
Substances
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Heat-Shock Proteins
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Multiprotein Complexes
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Nuclear Proteins
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Reactive Oxygen Species
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Recombinant Proteins
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SESN1 protein, human
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SESN2 protein, human
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Mechanistic Target of Rapamycin Complex 1
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TOR Serine-Threonine Kinases