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. 2018 Jan;109(1):e23069.
doi: 10.1002/bip.23069. Epub 2017 Oct 27.

The Polypeptide Biophysics of Proline/Alanine-Rich Sequences (PAS): Recombinant Biopolymers With PEG-like Properties

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Free PMC article

The Polypeptide Biophysics of Proline/Alanine-Rich Sequences (PAS): Recombinant Biopolymers With PEG-like Properties

Joscha Breibeck et al. Biopolymers. .
Free PMC article

Abstract

PAS polypeptides comprise long repetitive sequences of the small L-amino acids proline, alanine and/or serine that were developed to expand the hydrodynamic volume of conjugated pharmaceuticals and prolong their plasma half-life by retarding kidney filtration. Here, we have characterized the polymer properties both of the free polypeptides and in fusion with the biopharmaceutical IL-1Ra. Data from size exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy and quantification of hydrodynamic and polar properties demonstrate that the biosynthetic PAS polypeptides exhibit random coil behavior in aqueous solution astonishingly similar to the chemical polymer poly-ethylene glycol (PEG). The solvent-exposed PAS peptide groups, in the absence of secondary structure, account for strong hydrophilicity, with negligible contribution by the Ser side chains. Notably, PAS polypeptides exceed PEG of comparable molecular mass in hydrophilicity and hydrodynamic volume while exhibiting lower viscosity. Their uniform monodisperse composition as genetically encoded polymers and their biological nature, offering biodegradability, render PAS polypeptides a promising PEG mimetic for biopharmaceutical applications.

Keywords: PASylation; biomimetics; hydrodynamic volume; recombinant polypeptide; viscosity.

Figures

Figure 1
Figure 1
PAS polypeptides and their analysis by SDS‐PAGE. (A) Amino acid repeat sequences of PAS polypeptides investigated in this study and their percentages of Pro content. (B) SDS‐PAGE (12% (w/v) polyacrylamide gel60 stained with Coomassie Brilliant Blue) of various PAS‐IL‐1Ra fusion proteins. (C) Isolated PAS polypeptides, obtained after cleavage of TrxA‐PAS fusion proteins, in comparison with PEG polymers, both stained with BaI2 (7% (w/v) polyacrylamide stacking gel and 20% (w/v) running gel, borderline highlighted)
Figure 2
Figure 2
Hydrodynamic and shape properties of PAS‐IL‐1Ra fusion proteins as well as isolated PAS polypeptides investigated by SEC and DLS. (A) Chromatogram of IL‐1Ra and corresponding PAS fusion proteins with ∼200 residues comprising different PAS sequences. (B) Chromatogram of isolated (unmodified) PAS polypeptides in comparison with fluorescein‐labeled PEG polymers with varying lengths. (C) Exemplary rH‐correlogram (output of the DLS instrument software) for P/A#1(600) measured in triplicate by DLS, revealing monodispersity with narrow rH‐distribution. (D) Shape estimation of PAS polypeptides based on parameters from viscometry and DLS (see text and Supporting Information Methods). The P1A3 polypeptide and also the PEG polymers most closely match the shape of a sphere with an ideal axis ratio p = 1. With rising Pro content, the PAS polypeptides adopt more elongated structures corresponding to increased axis ratios. The PAS#1 and P/A#1 polypeptides with p = 5 are surpassed by the P1A1 polypeptide (p = 10, not shown), which exhibits PPII‐like structure
Figure 3
Figure 3
Viscosity of PAS polypeptides in comparison with PEG in aqueous solution. (A) Plots of viscosity against the sample concentration for PAS#1, P/A#1 and PEG with varied lengths at 25°C (fitted by a polynomial function, see Supporting Information Methods). (B) Evaluation of the Mark‐Houwink parameters from [η] = K · Ma for the three different polymers shown in (A). (C) Plot of [η] against the calculated hydrodynamic radius rHVisc for the three polymers. The intrinsic viscosity of both PAS polypeptides versus hydrodynamic volume shows almost the same slope, whereas PEG exhibits higher viscosity for similar rHVisc. (D) Mark‐Houwink‐Sakurada parameters evaluated from viscosity and SEC data using 3 different mathematical approaches (see text and Supporting Information Methods)
Figure 4
Figure 4
CD spectroscopy of PAS polypeptides. (A) CD spectra of purified PAS polypeptides. The characteristic wavelength window between 195 and 205 nm is indicated by dashed black lines, band minima (or maxima) are marked by black dots. (B) Difference CD spectra obtained for the IL‐1Ra fusion proteins after subtraction of the Il‐1Ra spectrum. (C) CD spectra (solid lines) of P/A#1(200), P/A#1(600) and PAS#1(600) at 20°C (set to ‐100%) and 90°C, together with the difference spectra between both temperatures (dashed lines). Independent of the length, all PAS polypeptides lose contributions from random‐coil structure and gain PPII structure at elevated temperatures (cf. Supporting Information Table S4). (D) Relative change in molar ellipticity for P/A#1(200), P/A#1(600), PAS#1(600), and P1A1(200) upon heating from 20 to 90°C with a linear temperature gradient of 1 K/min monitored at 195 nm (fitted by a straight line). (E) Illustration of the impact of Pro proportion on the ensemble conformation of P/A polypeptides (for explanation, see text). Zero Pro content corresponds to pure poly‐L‐alanine, which is known to adopt α‐helical conformation. Beyond a critical proportion of Pro residues P/A polypeptides adopt a random coil conformation with a three‐dimensionally expanded, almost spherical average appearance. However, with rising Pro content the molecular shape becomes increasingly elongated and, finally, the pure poly‐L‐proline chain exhibits full PPII conformation in aqueous solution

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