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Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine.
- Source :
-
Journal of Industrial Microbiology & Biotechnology . Jul2012, Vol. 39 Issue 7, p1041-1048. 8p. - Publication Year :
- 2012
-
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) 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-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 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 13675435
- Volume :
- 39
- Issue :
- 7
- Database :
- Academic Search Index
- Journal :
- Journal of Industrial Microbiology & Biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 77057797
- Full Text :
- https://doi.org/10.1007/s10295-012-1105-1