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Rapid method for an enhanced recovery of biologically active human phospholipid scramblase1 from inclusion bodies
Published in Academic Press Inc.
2018
PMID: 29964029
Volume: 556
   
Pages: 104 - 111
Abstract
Human phospholipid scramblase 1 (hPLSCR1) is an ATP independent, Ca2+ dependent transmembrane protein mediating bidirectional translocation of phospholipids across the lipid bilayer but the mechanism of scrambling is unknown. Determination of the hPLSCR1 structure would help understand the mechanism and its multi-functional property. Recombinant hPLSCR1 forms inclusion bodies (IBs), when over-expressed in E. coli BL21 (DE3) and recovery of active hPLSCR1 from IBs, were time-consuming and resulted in low protein yield. This study aims to optimize and enhance the expression and purification of active recombinant hPLSCR1 by various strategies. Additives including stabilizers and detergents were added during cell lysis to improve the recovery of soluble hPLSCR1. Five E. coli strains, BL21 (DE3), C43 (DE3), Rosetta, BL21-CodonPlus-RP, and BL21 (DE3) pLysS were screened for maximum yield of soluble protein but localized in IBs. To recover hPLSCR1 from IBs, different additives were added of which, 0.3% N-lauroyl sarcosine (NLS) recovered ∼50% of bioactive hPLSCR1 from IBs. E. coli C43 (DE3) gave higher yields of purified protein (7.76 mg/g cell) followed by E. coli BL21 (DE3) pLysS (5.87 mg/g cell). This report describes a rapid and efficient method for solubilizing membrane proteins from inclusion bodies with a higher recovery. © 2018 Elsevier Inc.
About the journal
JournalAnalytical Biochemistry
PublisherAcademic Press Inc.
ISSN00032697
Open AccessNo
Concepts (39)
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    HUMAN PHOSPHOLIPID SCRAMBLASE 1
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    Imidazole
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    Protein
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    PROTEOLIPOSOME
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    SCRAMBLASE
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    Unclassified drug
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    PHOSPHOLIPID TRANSFER PROTEIN
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    PLSCR1 PROTEIN, HUMAN
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    Recombinant protein
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    Article
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    Cell inclusion
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    Circular dichroism
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    Comparative study
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    Controlled study
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    CYTOLYSIS
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    Densitometry
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    Enzyme purification
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    Escherichia coli
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    Incubation time
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    Molecular weight
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    Nonhuman
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    Polyacrylamide gel electrophoresis
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    Priority journal
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    Protein analysis
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    Protein expression
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    Protein secondary structure
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    Solubilization
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    Ultraviolet spectroscopy
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    Western blotting
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    Biosynthesis
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    Chemistry
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    Human
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    Isolation and purification
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    Metabolism
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    Protein stability
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    Humans
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    Inclusion bodies
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    PHOSPHOLIPID TRANSFER PROTEINS
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    Recombinant proteins