De Novo Design of Supercharged, Unfolded Protein Polymers, and Their Assembly into Supramolecular Aggregates

Kolbe, Anke; Mercato, Loretta L. del; Abbasi, Azhar Z.; Rivera-Gil, Pilar; Gorzini, Sekineh J.; Huibers, Willem; Poolman, Berend; Parak, Wolfgang J.; Herrmann, Andreas

doi:10.1002/marc.201000491

Cited by 46 publications

(65 citation statements)

References 32 publications

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“…One such example is elastin-like polypeptides (ELPs), which are based on a common pentameric repeat sequence (VPGVG) found in tropoelastin. Because of the material's inherent mechanical elasticity and inverse temperature-transition behaviors, ELPs have gained considerable attention for biotechnological and biomedical applications (37,38). Here, we take advantage of the flexibility of amino acid composition in ELPs by introducing glutamic acid at the fourth position of the repeat to transform ELP into unfolded and highly charged anionic polyelectrolytes (38).…”

Section: Resultsmentioning

confidence: 99%

“…Because of the material's inherent mechanical elasticity and inverse temperature-transition behaviors, ELPs have gained considerable attention for biotechnological and biomedical applications (37,38). Here, we take advantage of the flexibility of amino acid composition in ELPs by introducing glutamic acid at the fourth position of the repeat to transform ELP into unfolded and highly charged anionic polyelectrolytes (38). Thus, genetically encoded ELPs with 72 negative charges along the backbone (E72) and a variant functionalized with green fluorescent protein (GFP) as fusion, E72-GFP, were produced in Escherichia coli (SI Appendix, Section D).…”

Section: Resultsmentioning

confidence: 99%

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Thermotropic liquid crystals from biomacromolecules

Li¹,

Chen

Marcozzi³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Complexation of biomacromolecules (e.g., nucleic acids, proteins, or viruses) with surfactants containing flexible alkyl tails, followed by dehydration, is shown to be a simple generic method for the production of thermotropic liquid crystals. The anhydrous smectic phases that result exhibit biomacromolecular sublayers intercalated between aliphatic hydrocarbon sublayers at or near room temperature. Both this and low transition temperatures to other phases enable the study and application of thermotropic liquid crystal phase behavior without thermal degradation of the biomolecular components.L iquid crystals (LCs) play an important role in biology because their essential characteristic, the combination of order and mobility, is a basic requirement for self-organization and structure formation in living systems (1-3). Thus, it is not surprising that the study of LCs emerged as a scientific discipline in part from biology and from the study of myelin figures, lipids, and cell membranes (4). These and the LC phases formed from many other biomolecules, including nucleic acids (5, 6), proteins (7,8), and viruses (9, 10), are classified as lyotropic, the general term applied to LC structures formed in water and stabilized by the distinctly biological theme of amphiphilic partitioning of hydrophilic and hydrophobic molecular components into separate domains. However, the principal thrust and achievement of the study of LCs has been in the science and application of thermotropic materials, structures, and phases in which molecules that are only weakly amphiphilic exhibit LC ordering by virtue of their steric molecular shape, flexibility, and/or weak intermolecular interactions [e.g., van der Waals and dipolar forces (11)]. These characteristics enable thermotropic LCs (TLCs) to adopt a wide variety of exotic phases and to exhibit dramatic and useful responses to external forces, including, for example, the electro-optic effects that have led to LC displays and the portable computing revolution. This general distinction between lyotropic LCs and TLCs suggests there may be interesting possibilities in the development of biomolecular or bioinspired LC systems in which the importance of amphiphilicity is reduced and the LC phases obtained are more thermotropic in nature. Such biological TLC materials are very appealing for several reasons. Most biomacromolecules were extensively characterized in aqueous environments, but in TLC phases, their solvent-free properties and functions could be investigated in a state in which no or only traces of water are present. Water exhibits a high dielectric constant and has the ability to form hydrogen bonds, greatly influencing the structure and functions of biomacromolecules or compromising electronic properties such as charge transport (12-15). Indeed, anhydrous TLC systems containing glycolipids (16-19), ferritin (20), and polylysine have been reported (21-23). However, a general approach to fabricating TLCs based on nucleic acids, polypeptides, proteins, and protein assemblies of larg...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Thermotropic liquid crystals from biomacromolecules

Li¹,

Chen

Marcozzi³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…We have previously reported the fabrication of unfolded supercharged polypeptides designed with elastin-like polypeptide (ELP) as the basic sequence [21]. These materials can be positively and negatively "supercharged" in a straightforward manner by introducing a lysine or glutamic acid residue at the second position of the repetitive pentapeptide sequence (GVGVP) [21].…”

Section: Introductionmentioning

confidence: 99%

“…These materials can be positively and negatively "supercharged" in a straightforward manner by introducing a lysine or glutamic acid residue at the second position of the repetitive pentapeptide sequence (GVGVP) [21]. This enables us to tune the overall charge of the protein by simply adjusting the length of the tag while obviating modification of the protein itself.…”

Section: Introductionmentioning

confidence: 99%

Enhancing cellular uptake of GFP via unfolded supercharged protein tags

Pesce

Kolbe

et al. 2013

Biomaterials

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a b s t r a c tOne of the barriers to the development of protein therapeutics is effective delivery to mammalian cells. The proteins must maintain a careful balance of polar moieties to enable administration and distribution and hydrophobic character to minimize cell toxicity. Numerous strategies have been applied to this end, from appending additional cationic peptides to supercharging the protein itself, sometimes with limited success. Here we present a strategy that combines these methods, by equipping a protein with supercharged elastin-like polypeptide (ELP) tags. We monitored cellular uptake and cell viability for GFP reporter proteins outfitted with a range of ELP tags and demonstrated enhanced uptake that correlates with the number of positive charges, while maintaining remarkably low cytotoxicity and resistance to degradation in the cell. GFP uptake proceeded mainly through caveolae-mediated endocytosis and we observed GFP emission inside the cells over extended time (up to 48 h). Low toxicity combined with high molecular weights of the tag opens the way to simultaneously optimize cell uptake and pharmacokinetic parameters. Thus, cationic supercharged ELP tags show great potential to improve the therapeutic profile of protein drugs leading to more efficient and safer biotherapeutics.

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“…As the wall of PEM capsules is porous [44] this is generally no problem. Porosity depends for example on the used polyelectrolyte materials and the number of polyelectrolyte layers [22,45] . A bigger problem is keeping the ionsensitive fl uorophores inside the capsules.…”

Section: Fluorescent Nps For Barcoding Of Capsules Enabling Spatiallymentioning

confidence: 99%

Nanoparticle-functionalized microcapsules for in vitro delivery and sensing

2012

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Inorganic nanoparticles such as magnetic nanoparticles, fl uorescent quantum dots, and plasmonic nanoparticles can be used as building blocks for designing multifunctional systems based on polymeric capsules. The properties of the inorganic nanoparticles hereby are harnessed to provide additional functionality to the polymer capsules. Biological applications towards in vitro sensing and delivery are discussed. Examples will be given in which magnetic nanoparticles are used to direct capsules with magnetic fi eld gradients, colloidal quantum dots are used to identify capsules via the formation of optical barcodes, and gold nanoparticles are used as light-controlled heat-sources for opening capsules and releasing macromolecules from their cavity upon optical excitation. This demonstrates that combination of inorganic nanoparticles and organic/polymeric molecules as carrier matrices allow for tailoring multifunctional hybrid particles for practical applications.

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De Novo Design of Supercharged, Unfolded Protein Polymers, and Their Assembly into Supramolecular Aggregates

Cited by 46 publications

References 32 publications

Thermotropic liquid crystals from biomacromolecules

Thermotropic liquid crystals from biomacromolecules

Enhancing cellular uptake of GFP via unfolded supercharged protein tags

Nanoparticle-functionalized microcapsules for in vitro delivery and sensing

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