Photolithographic surface functionalization for spatio-temporally controlled protein immobilization

Exploiting the functional diversity of proteins for fundamental research and biotechnological applications requires their functional organization into micro- and nanostructures while preserving their functional integrity to the highest possible level. My PhD research aimed to establish generic techniques based on photolithography which could be used to control the spatial as well as temporal organization of recombinantly expressed proteins on surfaces. My thesis describes in detail four strategies that I developed for achieving this goal. In the first approach a photo-induced Fenton reaction was used to selectively destroy tris(nitrilotriacetic acid) (tris-NTA) moieties on a surface. UV-irradiation through a photomask allowed localized photo-destruction and targeting of His-tagged proteins to non-irradiated regions. Photo-destruction could also be achieved by scanning selected regions with the UV laser of a confocal laser scanning microscope (CLSM) thus allowing flexible creation and modification of protein patterns. The second strategy was based on the photosensitive nitroveratryloxycarbonyl (NVOC) protection group, which was used to cage amine groups on a surface. Sequential uncaging by UV-irradiation through a photomask followed by reactions with biotin and coenzyme A was used to pattern streptavidin and ybbR-tagged proteins into microstructures. In the third approach a photo-fragmentable Histidine peptide was used to block tris-NTA surfaces against binding of His-tagged proteins. UV-irradiation through a photomask or by using a UV laser in a CLSM cleaved the peptide into short fragments which quickly dissociated from the surface due to loss in multivalency. His-tagged proteins could be efficiently targeted into irradiated regions even from a complex cell lysate. Sequential uncaging and immobilization allowed the construction of multiplexed protein patterns with a high degree of temporal control. The fourth strategy used combined peptide tags comprising of a His-tag as well as a Halo- or ybbR-tag to achieve rapid covalent immobilization of recombinant fusion proteins on surfaces functionalized with specific ligands. In combination with a photo-fragmentable histidine peptide as described above, stable spatio-temporal organization of proteins carrying these combined tags was possible. The techniques developed in this thesis enabled the photolithographical micropatterning of recombinant proteins carrying specific peptide or protein tags on surfaces in a functional manner. Owing to the generic nature of immobilization strategies, coupled with the ease of patterning, highly versatile applications of these methods both in fundamental research as well as bio-technological and analytical applications can be envisioned

Stimuli-sensitive intrinsically disordered protein brushes.

Grafting polymers onto surfaces at high density to yield polymer brush coatings is a widely employed strategy to reduce biofouling and interfacial friction. These brushes almost universally feature synthetic polymers, which are often heterogeneous and do not readily allow incorporation of chemical functionalities at precise sites along the constituent chains. To complement these synthetic systems, we introduce a biomimetic, recombinant intrinsically disordered protein that can assemble into an environment-sensitive brush. This macromolecule adopts an extended conformation and can be grafted to solid supports to form oriented protein brushes that swell and collapse dramatically with changes in solution pH and ionic strength. We illustrate the value of sequence specificity by using proteases with mutually orthogonal recognition sites to modulate brush height in situ to predictable values. This study demonstrates that stimuli-responsive brushes can be fabricated from proteins and introduces them as a new class of smart biomaterial building blocks

Stimuli-sensitive intrinsically disordered protein brushes.

Grafting polymers onto surfaces at high density to yield polymer brush coatings is a widely employed strategy to reduce biofouling and interfacial friction. These brushes almost universally feature synthetic polymers, which are often heterogeneous and do not readily allow incorporation of chemical functionalities at precise sites along the constituent chains. To complement these synthetic systems, we introduce a biomimetic, recombinant intrinsically disordered protein that can assemble into an environment-sensitive brush. This macromolecule adopts an extended conformation and can be grafted to solid supports to form oriented protein brushes that swell and collapse dramatically with changes in solution pH and ionic strength. We illustrate the value of sequence specificity by using proteases with mutually orthogonal recognition sites to modulate brush height in situ to predictable values. This study demonstrates that stimuli-responsive brushes can be fabricated from proteins and introduces them as a new class of smart biomaterial building blocks

Quantitative Real-Time Imaging of Protein–Protein Interactions by LSPR Detection with Micropatterned Gold Nanoparticles

Localized surface plasmon resonance (LSPR) offers powerful means for sensitive label-free detection of protein–protein interactions in a highly multiplexed format. We have here established self-assembly and surface modification of plasmonic nanostructures on solid support suitable for quantitative protein–protein interaction analysis by spectroscopic and microscopic LSPR detection. These architectures were obtained by layer-by-layer assembly via electrostatic attraction. Gold nanoparticles (AuNP) were adsorbed on a biocompatible amine-terminated poly­(ethylene glycol) (PEG) polymer brush and further functionalized by poly-l-lysine graft PEG (PLL-PEG) copolymers. Stable yet reversible protein immobilization was achieved via tris­(nitrilotriacetic acid) groups incorporated into the PLL-PEG coating. Thus, site-specific immobilization of His-tagged proteins via complexed Ni­(II) ions was achieved. Functional protein immobilization on the surface was confirmed by real-time detection of LSPR scattering by reflectance spectroscopy. Association and dissociation rate constants obtained for a reversible protein–protein interaction were in good agreement with the data obtained by other surface-sensitive detection techniques. For spatially resolved detection, AuNP were assembled into micropatterns by means of photolithographic uncaging of surface amines. LSPR imaging of reversible protein–protein interactions was possible in a conventional wide field microscope, yielding detection limits of ∼30 protein molecules within a diffraction-limited surface area

High concentration formulation developability approaches and considerations

ABSTRACTThe growing need for biologics to be administered subcutaneously and ocularly, coupled with certain indications requiring high doses, has resulted in an increase in drug substance (DS) and drug product (DP) protein concentrations. With this increase, more emphasis must be placed on identifying critical physico-chemical liabilities during drug development, including protein aggregation, precipitation, opalescence, particle formation, and high viscosity. Depending on the molecule, liabilities, and administration route, different formulation strategies can be used to overcome these challenges. However, due to the high material requirements, identifying optimal conditions can be slow, costly, and often prevent therapeutics from moving rapidly into the clinic/market. In order to accelerate and derisk development, new experimental and in-silico methods have emerged that can predict high concentration liabilities. Here, we review the challenges in developing high concentration formulations, the advances that have been made in establishing low mass and high-throughput predictive analytics, and advances in in-silico tools and algorithms aimed at identifying risks and understanding high concentration protein behavior

Site-Specific Modulation of Charge Controls the Structure and Stimulus Responsiveness of Intrinsically Disordered Peptide Brushes

Intrinsically disordered proteins (IDPs) are an important and emerging class of materials for tailoring biointerfaces. While the importance of chain charge and resultant electrostatic interactions in controlling conformational properties of IDPs is beginning to be explored through in silico approaches, there is a dearth of experimental studies motivated toward a systematic study of these effects. In an effort to explore this relationship, we measured the conformations of two peptides derived from the intrinsically disordered neurofilament (NF) side arm domain: one depicting the wild-type sequence with four lysine–serine–proline repeats (KSP peptide) and another in which the serine residues were replaced with aspartates (KDP peptide), a strategy sometimes used to mimic phosphorylation. Using a variety of biophysical measurements including a novel application of scanning angle interference microscopy, we demonstrate that the KDP peptide assumes comparatively more expanded conformations in solution and forms significantly thicker brushes when immobilized on planar surfaces at high densities. In both settings, the peptides respond to changes in ambient ionic strength, with each peptide showing distinct stimulus-responsive characteristics. While the KDP peptide undergoes compaction with increasing ionic strength as would be expected for a polyampholyte, the KSP peptide shows biphasic behavior, with an initial compaction followed by an expanded state at a higher ionic strength. Together these results support the notion that modulation of charge on IDPs can regulate conformational and interfacial properties

Modulating surface density of proteins via caged surfaces and controlled light exposure

We demonstrate the possibility of tuning the degree of functionalization of a surface using photoactivatable chemistries and controlled light exposure. A photosensitive organosilane with a protected amine terminal group and a tetraethyleneglycol spacer was synthesized. A o-nitrobenzyl cage was used as the photoremovable group to cage the amine functionality. Surfaces with phototunable amine densities were generated by controlled irradiation of silica substrates modified with the photosensitive anchor. Protein layers with different densities could be obtained by successive coupling and assembly steps. Protein surface concentrations were quantified by reflectance interference. Our results demonstrate that the protein density correlates with the photogenerated ligand density. The density control was proved over four coupling steps (biotin, SAv, BTtris-NTA, MBP, or GFP), indicating that the interactions between underlying layer and soluble targets are highly specific and the immobilized targets at the four levels maintain their full functionality. Protein micropatterns with a gradient of protein density were also obtained. © 2011 American Chemical Society.Fil: Álvarez, Marta. Max-Planck-Institut fur Polymerforschung; AlemaniaFil: Alonso, José María. Max Planck Institut fur Metallforschung; AlemaniaFil: Filevich, Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Bhagawati, Maniraj. Universitat Osnabruck; AlemaniaFil: Etchenique, Roberto Argentino. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Piehler, Jacob. Universitat Osnabruck; AlemaniaFil: del Campo, Aránzazu. Max-Planck-Institut fur Polymerforschung; Alemani

Single Cell GFP-Trap Reveals Stoichiometry and Dynamics of Cytosolic Protein Complexes

We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level