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. 2022 Mar 14;23(3):1195-1204.
doi: 10.1021/acs.biomac.1c01466. Epub 2022 Jan 18.

Charged Polypeptide Tail Boosts the Salt Resistance of Enzyme-Containing Complex Coacervate Micelles

Riahna Kembaren  1   2 , Adrie H Westphal  2 , Marleen Kamperman  3 , J Mieke Kleijn  1 , Jan Willem Borst  2
Affiliations

Charged Polypeptide Tail Boosts the Salt Resistance of Enzyme-Containing Complex Coacervate Micelles

Riahna Kembaren et al. Biomacromolecules. .

Abstract

Encapsulation of proteins can have advantages for their protection, stability, and delivery purposes. One of the options to encapsulate proteins is to incorporate them in complex coacervate core micelles (C3Ms). This can easily be achieved by mixing aqueous solutions of the protein and an oppositely charged neutral-hydrophilic diblock copolymer. However, protein-containing C3Ms often suffer from salt-inducible disintegration due to the low charge density of proteins. The aim of this study is to improve the salt stability of protein-containing C3Ms by increasing the net charge of the protein by tagging it with a charged polypeptide. As a model protein, we used CotA laccase and generated variants with 10, 20, 30, and 40 glutamic acids attached at the C-terminus of CotA using genetic engineering. Micelles were obtained by mixing the five CotA variants with poly(N-methyl-2-vinyl-pyridinium)-block-poly(ethylene oxide) (PM2VP128-b-PEO477) at pH 10.8. Hydrodynamic radii of the micelles of approximately 31, 27, and 23 nm for native CotA, CotA-E20, and CotA-E40, respectively, were determined using dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). The encapsulation efficiency was not affected using enzymes with a polyglutamic acid tail but resulted in more micelles with a smaller number of enzyme molecules per micelle. Furthermore, it was shown that the addition of a polyglutamic acid tail to CotA indeed resulted in improved salt stability of enzyme-containing C3Ms. Interestingly, the polyglutamic acid CotA variants showed an enhanced enzyme activity. This study demonstrates that increasing the net charge of enzymes through genetic engineering is a promising strategy to improve the practical applicability of C3Ms as enzyme delivery systems.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Comparison of electrostatic potentials at the molecular surface of three CotA variants. (A) Native CotA (CotA wild type), (B) CotA with additional 10 glutamic acid tags (CotA-E10), and (C) CotA with additional 20 glutamic acid tags (CotA-E20). Color surface overlay indicates the electrostatic potential in a scale from negative (red), neutral (white), and positive potential (blue). This figure was created using the default parameters of the PyMOL APBS Tools plugin in PyMol_2.3.4 software.
Figure 2
Figure 2
SDS-PAGE of purified CotA-WT, CotA-E10, CotA-E20, CotA-E30, and CotA-E40.
Figure 3
Figure 3
Light scattering intensity (I), hydrodynamic radius (Rh), and polydispersity index (PDI) for mixtures of different CotA variants with the diblock copolymer PM2PV128-b-PEO477 observed with DLS. (A) Native CotA, (B) CotA-E10, (C) CotA-E20, (D) CotA-E30, and (E) CotA-E40. The maximum in light scattering intensity as a function of F– corresponds to the preferred micellar composition (PMC).
Figure 4
Figure 4
Multiangle DLS results of C3M solutions containing (A) native CotA and (B) CotA-E40. The decay rate Γ obtained from the DLS correlation curves by a first (blue), second (orange), and third (gray) cumulant fit with squared wave vector q2.
Figure 5
Figure 5
Salt stability of enzyme-containing C3Ms was observed using DLS. (A) Normalized light scattering intensity (I), (B) hydrodynamic radius (Rh), and (C) polydispersity index (PDI). C3Ms composed of native CotA (blue), CotA-E20 (orange), and CotA-E40 (dark red). Error bars represent the standard deviation from three repetitions (n = 3).
Figure 6
Figure 6
FCS measurements on C3Ms composed of labeled enzymes. FCS autocorrelation curves (G(t)) for (A) native CotA (blue), (B) CotA-E20 (orange), and (C) CotA-E40 (dark red). The spheres represent the free enzyme, the triangles represent enzyme-containing C3Ms, and the squares represent enzyme-containing C3Ms with an additional 200 mM NaCl. (D) Normalized G(t) for free enzymes and C3M samples of native CotA and higher-charged CotA variants.
Figure 7
Figure 7
Salt stability of enzyme-containing C3Ms was observed using FCS. (A) Normalized number of fluorescent particles in the confocal volume (N) and (B) hydrodynamic radius (Rh). C3Ms composed of native CotA (blue), CotA-E20 (orange), and CotA-E40 (dark red). Error bars represent the standard deviation from three repetitions (n = 3).
Figure 8
Figure 8
Activity measurement of native CotA (CotA-WT), CotA-E10, CotA-E20, CotA-E30, and CotA-E40 and its C3Ms.
See this image and copyright information in PMC

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