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

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.

Microbial Lipases and Their Potential in the Production of Pharmaceutical Building Blocks

“…The excess of salts used for this purpose can be easily removed from the enzyme solution by techniques such as dialysis, ultrafiltration or size exclusion. In the case of hydrophobic ones, the use of detergents for their desorption is frequent, which makes it difficult to remove their excesses from the enzymatic solution by the techniques described, or where the use of polystyrene beads for this purpose is not an option since the lipase could also be co-immobilized on these [ 191 , 192 ]. However, the most outstanding advantage of supports that immobilize lipases by hydrophobic interaction is that they “mimic” the natural surface conditions in which lipases are usually more active ( Section 3.1.1 ).…”

A Tutorial Review on the Methodologies and Theories Utilized to Handle Proteins toward Obtaining Single Protein Crystals

“…Based on solubility , proteins usually encounter a slight increase in their solubility in the presence of small amounts of salts (so-called salting in ), but this solubility can drop drastically at increasing concentrations of salts (so-called salting out ), resulting in protein precipitation. , Since every protein has a specific salt type and concentration that can induce precipitation, salting out is widely used to fractionate proteins from one another and concentrate diluted protein solutions . Here comes the importance of dialysis (through a semipermeable membrane) as an essential method to purify the resulting protein fraction from the excess salt concentration and other small molecules and, if necessary, change its buffer and/or pH value to others favoring the stability of the protein in its active form. − Relying on the size and more precisely, the hydrodynamic radius of proteins, further purification of proteins can be realized using size-exclusion chromatography (also known as gel-filtration and molecular exclusion chromatography) . In this purification method, the protein fraction is placed on the top of a column containing porous beads (≈100 μm in diameter) made of highly hydrated yet insoluble polymer (commercially known as Sephadex, Sepharose, and Biogel).…”