title: Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine.
abstract: Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca(2+)) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca(2+) ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques.
authors.affiliation: Applied Industrial Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai, 600036, India.
authors.forename: Vincent Gerard
authors.initials: VG
authors.lastname: Francis
authors.forename: Mohammed Abdul
authors.initials: MA
authors.lastname: Majeed
authors.forename: Sathyanarayana N
authors.initials: SN
authors.lastname: Gummadi
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry; D014364:Tryptophan / Q000032:analysis
chemical_list: D008565:Membrane Proteins; C493422:PLSCR1 protein, human; D049294:Phospholipid Transfer Proteins; D011994:Recombinant Proteins; C025231:sarkosyl; D014364:Tryptophan; D012521:Sarcosine