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

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          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.

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          Author and article information

          Journal
          J. Ind. Microbiol. Biotechnol.
          Journal of industrial microbiology & biotechnology
          Springer Nature America, Inc
          1476-5535
          1367-5435
          Jul 2012
          : 39
          : 7
          Affiliations
          [1 ] Applied Industrial Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai, 600036, India.
          Article
          10.1007/s10295-012-1105-1
          22389205
          c4f0b62a-a78e-4be7-9352-4387c6240c81
          History

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