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

  • Fermentation, Cell Culture and Bioengineering
  • Published:
Journal of Industrial Microbiology & Biotechnology

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 (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.

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Acknowledgments

This work is supported by a research grant from the Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi. Vincent Gerard Francis acknowledges CSIR for providing fellowship.

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Authors and Affiliations

  1. Applied Industrial Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai, 600036, India

    Vincent Gerard Francis, Mohammed Abdul Majeed & Sathyanarayana N. Gummadi

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  1. Vincent Gerard Francis
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  2. Mohammed Abdul Majeed
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  3. Sathyanarayana N. Gummadi
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Correspondence to Sathyanarayana N. Gummadi.

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Francis, V.G., Majeed, M.A. & Gummadi, S.N. Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine. J Ind Microbiol Biotechnol 39, 1041–1048 (2012). https://doi.org/10.1007/s10295-012-1105-1

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  • DOI: https://doi.org/10.1007/s10295-012-1105-1

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