Coarse-grained simulation of PEGylated and tethered protein devices at all experimentally accessible surface residues on ß-lactamase for stability analysis and comparison.

PEGylated and surface-tethered proteins are used in a variety of biotechnological applications, but traditional methods offer little control over the placement of the functionalization sites on the protein. Fortunately, recent experimental methods functionalize the protein at any location on the amino acid sequence, so the question becomes one of selecting the site that will result in the best protein function. This work shows how molecular simulation can be used to screen potential attachment sites for surface tethering or PEGylation. Previous simulation work has shown promise in this regard for a model protein, but these studies are limited to screening only a few of the surface-accessible sites or only considered surface tethering or PEGylation separately rather than their combined effects. This work is done to overcome these limitations by screening all surface-accessible functionalization sites on a protein of industrial and therapeutic importance (TEM-1) and to evaluate the effects of tethering and PEGylation simultaneously in an effort to create a more accurate screen. The results show that functionalization site effectiveness appears to be a function of super-secondary and tertiary structures rather than the primary structure, as is often currently assumed. Moreover, sites in the middle of secondary structure elements, and not only those in loops regions, are shown to be good options for functionalization-a fact not appreciated in current practice. Taken as a whole, the results show how rigorous molecular simulation can be done to identify candidate amino acids for functionalization on a protein to facilitate the rational design of protein devices.

The Effects of p-Azidophenylalanine Incorporation on Protein Structure and Stability.

Functionalization is often needed to harness the power of proteins for beneficial use but can cause losses to stability and/or activity. State of the art methods to limit these deleterious effects accomplish this by substituting an amino acid in the wild-type molecule into an unnatural amino acid, such as p-azidophenylalanine (pAz), but selecting the residue for substitution a priori remains an elusive goal of protein engineering. The results of this work indicate that all-atom molecular dynamics simulation can be used to determine whether substituting pAz for a natural amino acid will be detrimental to experimentally determined protein stability. These results offer significant hope that local deviations from wild-type structure caused by pAz incorporation observed in simulations can be a predictive metric used to reduce the number of costly experiments that must be done to find active proteins upon substitution with pAz and subsequent functionalization.

Parameterization of Unnatural Amino Acids with Azido and Alkynyl R-Groups for Use in Molecular Simulations.

Recent new methods to functionalize proteins at specific amino acid locations use unnatural amino acids that contain azido and alkynyl groups. This capability is unprecedented and enables the creation of site-specific protein devices. Because of the high specificity of these devices, many protein configurations are possible and in silico screens have shown promise in predicting optimal attachment site locations. Therefore, there is significant interest in improving current molecular dynamics (MD) models to include the unique chemistries of these linear moieties. This work uses the force field tool kit to obtain the bonded and nonbonded CHARMM parameters for small molecules that contain azido and alkynyl groups. Next, the reliability of these parameters is tested by running simulated MD analysis to prove that the modeled structures match those found in the literature and quantum theory. Finally, the protein MD simulation compares this parameter set with crystallographic data to give a greater understanding of unnatural amino acid influence on the protein structure.

Endotoxin-Free E. coli-Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production.

Approximately one third of protein therapeutics are produced in Escherichia coli, targeting a wide variety of diseases. However, due to immune recognition of endotoxin (a lipid component in the E. coli cell membrane), these protein products must be extensively purified before application to avoid adverse reactions such as septic shock. E. coli-based cell-free protein synthesis (CFPS), which has emerged as a promising platform for the development and production of enhanced protein therapeutics, provides a unique opportunity to remove endotoxins prior to protein expression due to its open environment and the absence of live cells. Pre-expression endotoxin removal from CFPS reagents could simplify downstream processing, potentially enabling on-demand production of unique protein therapeutics. Herein, three strategies for removing endotoxins from E. coli cell lysate are evaluated: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi cells. It is demonstrated that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields. Further, the first adaptation of ClearColi cells to prepare cell-free extract with high protein synthesis capability is demonstrated. Finally, production of the acute lymphoblastic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi extract is demonstrated.

Nanoreactors: Strategies to encapsulate enzyme biocatalysts in virus-like particles.

Enzyme-mediated biocatalysis is generally more selective and environmentally friendly and requires less energy than chemocatalysis. However, factors such as temperature, acidity and the presence of proteases can negate enzyme activity. Encapsulation in virus-like particles is one promising method to mitigate these difficulties. Encapsulation also can be used to create multi-reaction nanoreactors that increase process efficiency by isolating reaction intermediates. To successfully encapsulate enzymes, a variety of methods involving both non-covalent and covalent interactions have been developed. Here we review promising virus-like particle encapsulation strategies, their advantages and remaining challenges.

The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation.

Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow experimental screening candidates. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for experimental and simulation screening systems such as the one presented here.