Zwitterionic polymers for SMALP Technology

The isolation of membrane proteins (MPs) from cell membranes has traditionally been carried out by the interaction of the membranes with detergents. This method had significant shortcomings when it comes to functionality and stability of the MPs. More recently, it was discovered that poly(styrene-co-maleic acid) (SMA) is able to isolate MPs with a layer of native phospholipids, leading to improved functionality and stability of the MPs as described in US2012142861A1. Although the use of SMA has led to significant improvement in the isolation of MPs, there are still shortcomings of this technology, such as limited stability in the presence of divalent cations (Ca²⁺, Mg²⁺, etc), limited stability at low and high pH, low resolution separation in gel electrophoresis (SDS-PAGE), also known as “smearing”. These shortcomings have different origins, and a variety of changes to the polymer have been suggested to overcome individual shortcomings in separate systems.

What we claim in this invention is a generally applicable base polymer that can be modified in a variety of different ways that in general overcome more than one of the reported shortcomings simultaneously. The base polymer is an alternating copolymer of styrene and maleic anhydride, partially modified into its N-benzylmaleimide derivative, where the alternating copolymer of styrene and maleic anhydride has a narrow molecular weight distribution (Ð < 1.3). The remaining maleic anhydride repeat units are subsequently modified in one of three different ways:

1. Hydrolysis into maleic acid repeat units
2. Conversion into a tertiary amine functional N-alkyl maleimide, such as structure (i)
3. Conversion into a zwitterionic functional N-alkyl maleimide, such as structure (ii)

In the isolation of membrane proteins, each one of the modified polymers gives a high-resolution separation on SDS-PAGE (little smearing). In addition, modification 1) leads to a polymer that provides stability at 5 < pH < 10 and [Mg²⁺] £ 20 mM. Modification 2) leads to a polymer that provides stability at 5 < pH < 8 and [Mg²⁺] > 100 mM. Modification 3) leads to a polymer that provides stability at 3 < pH < 10 and [Mg²⁺] > 100 mM.

The existing poly(styrene-co-maleic acid) (SMA) copolymer, has a maleic acid unit possessing two carboxylic acid groups, which undergo partial deprotonation at/around neutral pH. The negatively charged carboxylate groups renders SMA its hydrophilicity. However, at acidic condition (pH < 6) the both carboxylic acid groups are protonated resulting in the SMA copolymer becoming more hydrophobic, and forming a precipitate. Additionally, in the presence of divalent cations at high concentrations (> 5 mM), the two carboxylic acid groups of SMA chelate with the M2+ cation resulting in aggregation. The pH and ionic instability of SMA is problematic when dealing with membrane systems that only solubilize at low pH conditions, or membrane proteins that participate in biochemical reactions that require the presence of divalent metal cations (e.g. Ca2+, Mg2+, etc.). To this end, the use of PCZ2K copolymer demonstrated enhanced pH stability (from 4 – 10) and increased resistance to high concentrations of MgCl2 salt (0 – 50 mM).  

In addition to the improved pH and ionic stability, the PCZ2K copolymer also demonstrated the ability to isolate a model protein, i.e., ZipA, with high purity and high concentration, and this behavior was comparable to commercially available SMA2000P variant with a similar polymer backbone.  

Prof Lubertus Klumperman, Faculty of Science, Department of Chemistry and Polymer Science

Dr Reuben Pfukwa, Faculty of Science, Department of Chemistry and Polymer Science

Sithando Sibariboyi, Faculty of Science, Department of Chemistry and Polymer Science