Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine

• Published in: Journal of industrial microbiology & biotechnology 2012-07, Vol.39 (7), p.1041-1048
• Main Authors: Francis, Vincent Gerard, Majeed, Mohammed Abdul, Gummadi, Sathyanarayana N
• Format: Article
• Language: English
• Subjects: Acids; affinity chromatography; Biochemistry; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; calcium; Cell Culture and Bioengineering; Chromatography; Chromatography, Affinity; Chromatography, Gel; Circular Dichroism; E coli; Escherichia coli; Escherichia coli - cytology; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Fluorescence; Fundamental and applied biological sciences. Psychology; Genetic Engineering; Humans; inclusion bodies; Inclusion Bodies - chemistry; Inclusion Bodies - enzymology; Inorganic Chemistry; Life Sciences; Lipids; membrane proteins; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Microbiology; Molecular weight; Nickel; Nitrilotriacetic acid; Phospholipid Transfer Proteins - chemistry; Phospholipid Transfer Proteins - genetics; Phospholipid Transfer Proteins - isolation & purification; Phospholipid Transfer Proteins - metabolism; phospholipids; plasma membrane; Protein Folding; protein secondary structure; Protein Structure, Secondary; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sarcosine - analogs & derivatives; Sarcosine - chemistry; Tryptophan - analysis
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-012-1105-1

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 (Ca2+) 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 (Ni2+–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 Ca2+ 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.