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

Santosh Kumar Palanirajan,Sathyanarayana N Gummadi,Punitha Govindasamy

doi:10.1038/s41598-020-73522-1

Santosh Kumar Palanirajan, Sathyanarayana N Gummadi + Show 1 more

Open Access

https://doi.org/10.1038/s41598-020-73522-1

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Journal: Scientific Reports	Publication Date: Oct 1, 2020
Citations: 3	License type: open-access

Affiliation: Indian Institute of Technology Madras

Abstract

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.

Highlights

Membrane protein purification is a laborious, expensive, and protracted process involving detergents for its extraction
We have identified that N-Lauryl sarcosine (NLS), a mild anionic detergent improved the recovery of human phospholipid scramblases (hPLSCRs) from inclusion bodies (IBs) to 50% at a concentration of 0.3% in 4 h19
The N-lauryl sarcosine (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

Summary

Introduction

Membrane protein purification is a laborious, expensive, and protracted process involving detergents for its extraction. 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. 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. A higher concentration of detergents is generally required to extract the MPs and the presence of excess detergent could potentially affect the stability or interfere with further purification techniques. 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. S-1)

Methods

Results

Conclusion