Selective Labeling and Decoration of the Ends and Sidewalls of Single-Walled Carbon Nanotubes Using Mono- and Bispecific Solid-Binding Fluorescent Proteins.

Simple and robust strategies for the noncovalent functionalization of carbon nanostructures with proteins are of considerable interest in hybrid nanomaterials synthesis, part-to-part assembly, and biosensor development. Here, we show that fusion of the Car9 and Car15 carbon-binding peptides to the C-termini of the sfGFP and mCherry fluorescent proteins enables selective labeling of the ends or the sidewalls of single walled carbon nanotubes. By installing a gold-binding peptide or a single cysteine residue in carbon-binding variants of sfGFP, we further produce heterobifunctional solid-binding proteins that support the decoration of nanotubes sidewalls or termini with gold nanoparticles. The approach described here is generic and should prove useful for the controlled assembly of other hybrid materials.

Affinity purification of Car9-tagged proteins on silica matrices: Optimization of a rapid and inexpensive protein purification technology.

Car9, a dodecapeptide identified by cell surface display for its ability to bind to the edge of carbonaceous materials, also binds to silica with high affinity. The interaction can be disrupted with l-lysine or l-arginine, enabling a broad range of technological applications. Previously, we reported that C-terminal Car9 extensions support efficient protein purification on underivatized silica. Here, we show that the Car9 tag is functional and TEV protease-excisable when fused to the N-termini of target proteins, and that it supports affinity purification under denaturing conditions, albeit with reduced yields. We further demonstrate that capture of Car9-tagged proteins is enhanced on small particle size silica gels with large pores, that the concomitant problem of nonspecific protein adsorption can be solved by lysing cells in the presence of 0.3% Tween 20, and that efficient elution is achieved at reduced l-lysine concentrations under alkaline conditions. An optimized small-scale purification kit incorporating the above features allows Car9-tagged proteins to be inexpensively recovered in minutes with better than 90% purity. The Car9 affinity purification technology should prove valuable for laboratory-scale applications requiring rapid access to milligram-quantities of proteins, and for preparative scale purification schemes where cost and productivity are important factors.

EKylation: Addition of an Alternating-Charge Peptide Stabilizes Proteins.

For nearly 40 years, therapeutic proteins have been stabilized by chemical conjugation of polyethylene glycol (PEG), but recently zwitterionic materials have proved to be a more effective substitute. In this work, we demonstrate that genetic fusion of alternating-charge extensions consisting of anionic glutamic acid (E) and cationic lysine (K) is an effective strategy for protein stabilization. This bioinspired "EKylation" method not only confers the stabilizing benefits of poly(zwitterions) but also allows for rapid biosynthesis of target constructs. Poly(EK) peptides of different predetermined lengths were appended to the C-terminus of a native ß-lactamase and its destabilized TEM-19 mutant. The EK-modified enzymes retained biological activity and exhibited increased stability to environmental stressors such as high temperature and high-salt solutions. This one-step strategy provides a broadly applicable alternative to synthetic polymer conjugation that is biocompatible and degradable.

A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.

We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

Carbon-binding designer proteins that discriminate between sp2- and sp3-hybridized carbon surfaces.

Robust and simple strategies to directly functionalize graphene- and diamond-based nanostructures with proteins are of considerable interest for biologically-driven manufacturing, biosensing, and bioimaging. Here, we identify a new set of carbon-binding peptides that vary in overall hydrophobicity and charge and engineer two of these sequences (Car9 and Car15) within the framework of E. coli thioredoxin 1 (TrxA). We develop purification schemes to recover the resulting TrxA derivatives in a soluble form and conduct a detailed analysis of the mechanisms that underpin the interaction of the fusion proteins with carbonaceous surfaces. Although equilibrium quartz crystal microbalance measurements show that TrxA::Car9 and TrxA::Car15 have similar affinities for sp(2)-hybridized graphitic carbon (Kd = 50 and 90 nM, respectively), only the latter protein is capable of dispersing carbon nanotubes. Further investigation by surface plasmon resonance and atomic force microscopy reveals that TrxA::Car15 interacts with sp(2)-bonded carbon through a combination of hydrophobic and π-π interactions but that TrxA::Car9 exhibits a cooperative mode of binding that relies on a combination of electrostatics and weaker π stacking. Consequently, we find that TrxA::Car9 binds equally well to sp(2)- and sp(3)-bonded (diamondlike) carbon particles whereas TrxA::Car15 is capable of discriminating between the two carbon allotropes. Our results emphasize the importance of understanding both bulk and molecular recognition events when exploiting the adhesive properties of solid-binding peptides and proteins in technological applications.

Direct and reversible immobilization and microcontact printing of functional proteins on glass using a genetically appended silica-binding tag.

Fusion of disulfide-constrained or linear versions of the Car9 dodecapeptide to model fluorescent proteins support their on-contact and oriented immobilization onto unmodified glass. Bound proteins can be released and the surface regenerated by incubation with l-lysine. This noncovalent chemistry enables rapid and reversibe microcontact printing of tagged proteins and speeds up the production of bicontinuous protein patterns.