Potent Inhibition of Ribonuclease A by Oligo(vinylsulfonic Acid)

Smith, Bryan D.; Soellner, Matthew B.; Raines, Ronald T.

doi:10.1074/jbc.m301852200

Cited by 64 publications

(63 citation statements)

References 44 publications

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“…We caution, however, that several synthetic polyanions are known to be potent inhibitors of RNase A. 63 Our initial experiments suggest that the presence of the SPS in these solutions can act to inhibit the activity of RNase A in these assays, and thus additional analytical experiments will be required to establish quantitatively the activity of the RNase A released from these materials. Second, although the RNase A-R 9 /SPS films investigated here dissolve and release their contents rapidly, it may prove possible to incorporate new polymer structures or other design elements that permit gradual erosion and the sustained release of protein.…”

Section: Surface-mediated Delivery Of Rnase A-r 9 To Cellsmentioning

confidence: 96%

Multilayered Films Fabricated from an Oligoarginine-Conjugated Protein Promote Efficient Surface-Mediated Protein Transduction

et al. 2007

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The conjugation of cationic protein transduction domains to proteins results in an increase in the extent to which proteins are internalized by cells. This investigation sought to determine whether the conjugation of a protein transduction domain to a functional protein could be used to facilitate the incorporation of the protein into multilayered polyelectrolyte films and, subsequently, whether these films could be used to promote surface-mediated protein transduction. We demonstrate that it is possible to fabricate multilayered assemblies 80 nm thick using sodium polystyrene sulfonate (SPS) and bovine pancreatic ribonuclease (RNase A) conjugated to the cationic protein transduction domain nonaarginine (R 9 ) using an entirely aqueous layer-by-layer process. We demonstrate further that the conjugation of R 9 to RNase A permits the assembly of multilayered films under conditions that do not allow for the incorporation of the unmodified protein. This result suggests that R 9 functions as a cationic anchor and serves to increase the strength of electrostatic interactions with SPS and facilitate layer-by-layer assembly. We also demonstrate that RNase A-R 9 /SPS films dissolve rapidly in physiologically relevant media and that macroscopic objects coated with these materials can be used to mediate high levels of protein transduction in mammalian cells. These results suggest the basis of general methods that could contribute to the design of materials that permit spatial and temporal control over the delivery of therapeutic proteins to cells and tissues.

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Section: Surface-mediated Delivery Of Rnase A-r 9 To Cellsmentioning

confidence: 96%

Multilayered Films Fabricated from an Oligoarginine-Conjugated Protein Promote Efficient Surface-Mediated Protein Transduction

et al. 2007

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“…It has been reported that PVS is a potent RNase A inhibitor (Smith et al, 2003). An RNase A inhibition assay was developed by modifying Ambion's RNase Alert Kit to quantify levels of PVS in purification pools.…”

Section: Analytical Techniquesmentioning

confidence: 98%

Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

McDonald

Victa

Carter-Franklin

et al. 2008

Biotech & Bioengineering

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We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody-polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to < 1 microg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody.

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“…Protein purification columns were from GE Biosciences (Piscataway, NJ). MES buffer (Sigma-Aldrich, St. Louis, MO) was purified by anion-exchange chromatography to remove trace amounts of oligomeric vinylsulfonic acid (37). Phosphatebuffered saline (PBS) contained (in 1.00 L) NaCl (8.0 g), KCl (2.0 g), Na 2 HPO 4 ·7H 2 O (1.15 g), KH 2 PO 4 (2.0 g), and NaN 3 (0.10 g) and had a pH of 7.4.…”

Section: Experimental Procedures Materialsmentioning

confidence: 99%

Cytotoxic Ribonucleases: The Dichotomy of Coulombic Forces

et al. 2007

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Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steadystate level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t 1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.Cells control their intracellular environment through careful gating of the influx of extracellular material (1,2). To distinguish between molecules to be internalized from those to be excluded, cells use specific interactions with cell-surface proteins, lipids, and carbohydrates. Nonspecific interactions mediated by Coulombic forces can also lead to internalization, often in an unregulated manner (1-5). 1 Such nonspecific interactions can be modulated by increasing, decreasing, or masking the cationic charge on the biomolecule (3,5).Two classes of molecules that exploit high cationicity to effect nonspecific adsorption to the cell surface and internalization are cell-penetrating peptides (CPPs 2 ) and ribonucleases (7-10). CPPs have received considerable attention due to their ability to transport otherwise membrane-impermeable cargo into cells (9,11,12). The detailed mechanism of CPP internalization is unclear, but is known to involve multiple steps. Those steps include binding † This work was supported by Grant CA73808 (NIH). R.J.J. and L.D.L. were supported by Biotechnology Training grant 08349 (NIH).L.D.L. was also supported by an ACS Division of Organic Chemistry Fellowship, sponsored by the Genentech Foundation. The Biophysics Instrumentation Facility was established with grants BIR-9512577 (NSF) and RR13790 (NIH).*To whom correspondence should be addressed: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544. Telephone: 608-262-8588. Fax: 608-262-3453. raines@biochem.wisc.edu.. 1 We prefer to use the term Coulombic rather than electrostatic to describe the for...

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Potent Inhibition of Ribonuclease A by Oligo(vinylsulfonic Acid)

Cited by 64 publications

References 44 publications

Multilayered Films Fabricated from an Oligoarginine-Conjugated Protein Promote Efficient Surface-Mediated Protein Transduction

Multilayered Films Fabricated from an Oligoarginine-Conjugated Protein Promote Efficient Surface-Mediated Protein Transduction

Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

Cytotoxic Ribonucleases: The Dichotomy of Coulombic Forces

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