The polypeptide biophysics of proline/alanine‐rich sequences (PAS): Recombinant biopolymers with PEG‐like properties

Breibeck, Joscha; Skerra, Arne

doi:10.1002/bip.23069

Cited by 50 publications

(54 citation statements)

References 55 publications

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“…polypeptides constitute highly water-soluble biosynthetic polymers (Breibeck & Skerra, 2018); for example, PA200 can be readily dissolved in aqueous buffers at concentrations of up to 50%(w/v), corresponding to 31 mM. The molecular mass of PA200 (16 225 Da) is 4.8-fold higher than the reference PEG polymer PEG 3350 applied in this study, whereas its length in a fully extended conformation exceeds that of PEG 3350 by a factor of just 2.6.…”

Section: Resultsmentioning

confidence: 86%

“…Taken together, we have demonstrated the utility of a PAS polypeptide as a precipitant to grow protein crystals, adding this novel class of biopolymers to the toolset for protein crystallography. Variations in length and PAS sequence as well as amino-acid composition, as described previously (Breibeck & Skerra, 2018;Schlapschy et al, 2013), may offer parameters for its future optimization as a novel type of protein crystallization reagent.…”

Section: Resultsmentioning

confidence: 99%

“…1), exhibit physicochemical properties that are surprisingly similar to those of PEG. Notably, PAS polypeptides of comparable mass show an increased hydrophilicity and hydrodynamic volume but a lower viscosity than PEGs (Breibeck & Skerra, 2018). Moreover, PAS polymers have no net charge and do not affect the pH of aqueous solutions.…”

Section: Introductionmentioning

confidence: 99%

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Proline/alanine-rich sequence (PAS) polypeptides as an alternative to PEG precipitants for protein crystallization

Schiefner¹,

Walser²,

Gebauer³

et al. 2020

Acta Crystallogr F Struct Biol Commun

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Proline/alanine-rich sequence (PAS) polypeptides represent a novel class of biosynthetic polymers comprising repetitive sequences of the small proteinogenic amino acids L-proline, L-alanine and/or L-serine. PAS polymers are strongly hydrophilic and highly soluble in water, where they exhibit a natively disordered conformation without any detectable secondary or tertiary structure, similar to polyethylene glycol (PEG), which constitutes the most widely applied precipitant for protein crystallization to date. To investigate the potential of PAS polymers for structural studies by X-ray crystallography, two proteins that were successfully crystallized using PEG in the past, hen egg-white lysozyme and the Fragaria × ananassa O-methyltransferase, were subjected to crystallization screens with a 200-residue PAS polypeptide. The PAS polymer was applied as a precipitant using a vapor-diffusion setup that allowed individual optimization of the precipitant concentration in the droplet in the reservoir. As a result, crystals of both proteins showing high diffraction quality were obtained using the PAS precipitant. The genetic definition and precise macromolecular composition of PAS polymers, both in sequence and in length, distinguish them from all natural and synthetic polymers that have been utilized for protein crystallization so far, including PEG, and facilitate their adaptation for future applications. Thus, PAS polymers offer potential as novel precipitants for biomolecular crystallography.

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Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

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Proline/alanine-rich sequence (PAS) polypeptides as an alternative to PEG precipitants for protein crystallization

Schiefner¹,

Walser²,

Gebauer³

et al. 2020

Acta Crystallogr F Struct Biol Commun

Self Cite

View full text Add to dashboard Cite

show abstract

“…A promising biological alternative to PEG is PAS: PAS is a heteropolymeric amino acid sequence incorporating the three monomers proline, alanine and serine (Schlapschy et al, 2013). These amino acids adopt an unfolded disordered conformation if assembled in a suitable manner, thus exposing the hydrophilic polypeptide backbone (with only a minor contribution of the serine hydroxy group to the overall hydrophilicity) (Breibeck and Skerra, 2018). One PAS unit consists of ∼20 monomers, and multiple PAS repeats can be chained to increase the shielding effect.…”

Section: Alternatives To Pegmentioning

confidence: 99%

“…The resulting polymeric sequence has biophysical properties which are similar to those of PEG, conferring an increased hydrodynamic radius on its fusion partners. As with PEG, this property varies with chain length, and a 400 residue PAS sequence is approximately equivalent to a 30 kDa linear PEG chain in its elution characteristics from a size exclusion matrix (Breibeck and Skerra, 2018).…”

Section: Alternatives To Pegmentioning

confidence: 99%

Overcoming Physiological Barriers to Nanoparticle Delivery—Are We There Yet?

Thomas

Weber

2019

Front. Bioeng. Biotechnol.

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The exploitation of nanosized materials for the delivery of therapeutic agents is already a clinical reality and still holds unrealized potential for the treatment of a variety of diseases. This review discusses physiological barriers a nanocarrier must overcome in order to reach its target, with an emphasis on cancer nanomedicine. Stages of delivery include residence in the blood stream, passive accumulation by virtue of the enhanced permeability and retention effect, diffusion within the tumor lesion, cellular uptake, and arrival at the site of action. We also briefly outline strategies for engineering nanoparticles to more efficiently overcome these challenges: Increasing circulation half-life by shielding with hydrophilic polymers, such as PEG, the limitations of PEG and potential alternatives, targeting and controlled activation approaches. Future developments in these areas will allow us to harness the full potential of nanomedicine.

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Protein Engineering Strategies for Improved Pharmacokinetics

Rondon

Mahri

Morales-Yánez

et al. 2021

Adv Funct Materials

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Protein therapeutics have gained momentum in recent years and become a pillar in treating many diseases and the only choice in several ailments. Protein therapeutics are highly specific, tunable, and less toxic than conventional small drug molecules. However, reaping the full benefits of therapeutic proteins in the clinics is often hindered by issues of immunogenicity and short half-life due essentially to fast renal clearance and enzymatic degradation. Advances in polymer chemistry and protein engineering allowed overcoming some of these limitations. Strategies to prolong the half-life of proteins rely on increasing their size and stability and/or fusing them to endogenous proteins (albumin, Fc fragment of antibody) to hijack physiological pathways involved in protein recycling. On the downside, these modifications might alter therapeutic proteins structure and function. Therefore, a compromise between half-life and activity is sought. This review covers half-life extension strategies using natural and synthetic polymers as well as fusion to other proteins and sheds light on genetic engineering strategies and chemical and enzymatic reactions to achieve this goal. Promising strategies and successful applications in the clinics are highlighted. DOI: . /adfm.functions that cannot be mimicked by simple chemical compounds. Since the action of proteins is highly specific, they barely interfere with normal biological processes and cause less adverse events. Protein therapeutics are frequently derived from proteins naturally produced by the body. These agents are therefore often well tolerated and poorly immunogenic. However, proteins also suffer from significant limitations. Proteins with a molecular weight below the threshold for kidney filtration ( kDa, the size of human serum albumin) are cleared from the systemic circulation within a day. Many proteins are even cleared within a few hours or a few minutes when metabolism contributes to elimination. Therefore, therapeutic proteins need to be injected to patients several times a week (e.g., erythropoietin) or even several times a day (e.g., glucagon-like peptide-or GLP-), resulting in peaks and valleys in plasma concentrations with the alternate risks of systemic side effects and suboptimal therapeutic concentrations. Moreover, frequent administration of medication causes patient discomfort and reduces quality of life. A second limitation of proteins lies in protein immunogenicity. Foreign proteins from prokaryotes or animals might present interesting therapeutic properties in humans. However, intrinsic immunogenicity of nonhuman proteins hampers their therapeutic use in the clinic because specific antibodies generated against the foreign protein neutralize its activity and result in a loss of therapeutic efficacy over time. The unwanted immune response might even cause more serious general immune effects such as anaphylaxis.Over the last three decades, protein engineering has largely demonstrated that it can provide solutions to the limitations of natural proteins. B...

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The polypeptide biophysics of proline/alanine‐rich sequences (PAS): Recombinant biopolymers with PEG‐like properties

Cited by 50 publications

References 55 publications

Proline/alanine-rich sequence (PAS) polypeptides as an alternative to PEG precipitants for protein crystallization

Proline/alanine-rich sequence (PAS) polypeptides as an alternative to PEG precipitants for protein crystallization

Overcoming Physiological Barriers to Nanoparticle Delivery—Are We There Yet?

Protein Engineering Strategies for Improved Pharmacokinetics

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