Rapid method for an enhanced recovery of biologically active human phospholipid scramblase1 from inclusion bodies

Palanirajan, Santosh Kumar; Gummadi, Sathyanarayana N.

doi:10.1016/j.ab.2018.06.028

Cited by 4 publications

(8 citation statements)

References 28 publications

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“…The insoluble fraction was added with 0.3% N-Lauryl Sarcosine (NLS). The resuspended solution was agitated at 16 °C, 110 rpm for 4 h 19 . Post incubation the resuspended solution was subjected to centrifugation at for 30 min at 13,000×g and 4 °C.…”

Section: Solubilization Of Inclusion Bodies (Ibs)mentioning

confidence: 99%

“…The protein fraction was reconstituted into liposomes for functional assay and is termed proteoliposomes which were prepared similar to that of liposomes. To the dissolved lipid mixture, 100 µg of recombinant hPLSCR3 was added after the solubilization step and then detergent was slowly removed by Bio-Beads 19,21,22 .…”

Section: Purification Of Hplscr3mentioning

confidence: 99%

“…The labeled liposomes and proteoliposomes collected from the previous step were resuspended in 1 ml of assay buffer and were divided into two equal fractions. The divided fractions were incubated either in the presence of 3 mM EDTA or 3 mM Ca 2+ at 37 °C for 4 h 19,21 .…”

Section: Purification Of Hplscr3mentioning

confidence: 99%

“…Overexpression of hPLSCRs (6 × Histidine Tag) in E. coli resulted in localization of hPLSCRs to inclusion bodies (IBs). From our previous studies, we have identified that N-Lauryl sarcosine (NLS), a mild anionic detergent improved the recovery of hPLSCR from IBs to 50% at a concentration of 0.3% in 4 h 19 . The NLS treated fraction is subjected to immobilized metal affinity chromatography (IMAC) where His tag binding to Ni-NTA matrix is affected in the presence of NLS.…”

mentioning

confidence: 99%

“…The current study is focused on evaluating the efficacy of this strategy for the purification of membrane proteins as an alternate tool for conventional dialysis. Therefore to proceed further with the optimization, the recombinant 6 × His-hPLSCR3 cloned in pET28a( +) is used and the insoluble inclusion bodies are separated and solubilized with 0.3% NLS as reported earlier 19,21 . The soluble fraction was added with the Bio-Beads SM2 for detergent removal while the fraction subjected to conventional dialysis served as the control experiment ( Supplementary Fig.…”

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confidence: 99%

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Polystyrene adsorbents: rapid and efficient surrogate for dialysis in membrane protein purification

Palanirajan

Govindasamy

Gummadi

2020

Sci Rep

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Membrane protein purification is a laborious, expensive, and protracted process involving detergents for its extraction. Purifying functionally active form of membrane protein in sufficient quantity is a major bottleneck in establishing its structure and understanding the functional mechanism. Although overexpression of the membrane proteins has been achieved by recombinant DNA technology, a majority of the protein remains insoluble as inclusion bodies, which is extracted by detergents. Detergent removal is essential for retaining protein structure, function, and subsequent purification techniques. In this study, we have proposed a new approach for detergent removal from the solubilized extract of a recombinant membrane protein: human phospholipid scramblase 3 (hPLSCR3). N-lauryl sarcosine (NLS) has been established as an effective detergent to extract the functionally active recombinant 6X-his- hPLSCR3 from the inclusion bodies. NLS removal before affinity-based purification is essential as the detergent interferes with the matrix binding. Detergent removal by adsorption onto hydrophobic polystyrene beads has been methodically studied and established that the current approach was 10 times faster than the conventional dialysis method. The study established the potency of polystyrene-based beads as a convenient, efficient, and alternate tool to dialysis in detergent removal without significantly altering the structure and function of the membrane protein.

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Section: Solubilization Of Inclusion Bodies (Ibs)mentioning

confidence: 99%

Section: Purification Of Hplscr3mentioning

confidence: 99%

Section: Purification Of Hplscr3mentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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