Proteins in solution: Fractal surfaces in solutions

The concept of the surface of a protein in solution, as well of the interface between protein and 'bulk solution', is introduced. The experimental technique of small angle X-ray and neutron scattering is introduced and described briefly. Molecular dynamics simulation, as an appropriate computational tool for studying the hydration shell of proteins, is also discussed. The concept of protein surfaces with fractal dimensions is elaborated. We finish by exposing an experimental (using small angle X-ray scattering) and a computer simulation case study, which are meant as demonstrations of the possibilities we have at hand for investigating the delicate interfaces that connect (and divide) protein molecules and the neighboring electrolyte solution.Comment: 8 pages, 5 figure

Integrating Nanomembrane Separation with Plasmonic Detection for Real-Time Cell Culture Monitoring

To further understand cellular responses to drug treatment the dynamics of a reduced secretome shall be investigated. Currently there is no method for the detection of secreted small molecules in real time, label-free and with a high resolution. We present a novel design, which integrates nanopore filtration technology with highly sensitive plasmonic detection that allows real time monitoring of filtered molecules with a high spatial resolution and label free. The cell culture chamber is separated from the site of detection only by our biocompatible nanomembrane filter with a thickness of less than 100 nm to exclude the majority of background signals from the cell culture. The fast filtration of the cell culture constituents through the nanomembrane to the detector allows the observation of the dynamics of secreted molecules during cell culture and/or drug application. The setup offers new possibilities for drug screening and cell assays and may reveal new insights into cell signaling and drug responses. This setup shall be used to monitor cell culture or tissue culture without the necessity of labeling. This can be particularly important for the very popular “organ-on-a-chip” or “patient-on-a-chip” approaches to monitor tissue reactions to drug treatments with a high spatial resolution. Please click Additional Files below to see the full abstract

Branched Polyethylene Glycol for Protein Precipitation

Ph.DDOCTOR OF PHILOSOPH

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ: a study of an industrial mAb producing CHO-S cell line

There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs

High-throughput, parallelized and automated protein purification for therapeutic antibody development

Abstract Antibody therapeutic development often involves significant demands for purified protein samples, from initial assessments of numerous constructs from early stage screening campaigns through to lead identification and then for process development and pilot scale runs. Efforts to reduce timelines and cost per sample are common to both platform purification and for process development. In the earliest stages, high-throughput purification platforms that utilize liquid handlers or other small volume approaches can be suitable, as the quantity requirements for assays are minimal. However, as the number of candidate molecules diminishes, the scope of assays can quickly expand and include a variety of cell-based and in vivo experiments which can require tens or hundreds of milligrams of products of defined purity and with low endotoxin levels. Purification of these samples in a high-throughput, parallelized manner represents a significant challenge with relatively few available off-the-shelf solutions. Process development requirements are also amendable to high-throughput purification strategies combined with statistical approaches in order to optimize the design space and narrow initial process operation parameters suitable for a given purification unit operation. While less often utilized, non-chromatographic purification methods may also be amenable to automation and parallelization at the initial stages of purification development

Molecular simulation of adsorption from dilute solutions

Adsorption of biomolecules on surfaces is a perennial and general challenge relevant to many fields in biotechnology. A change of the Helmholtz free energy ΔA takes place when a molecule becomes adsorbed out of a bulk solution. The purpose of our investigations is to explore routes for the calculation of ΔA by molecular simulations. ΔA can be obtained both by integration over the mean force on a molecule and via the local density. It turns out that the route via the potential of mean force prevails over the latter due to better consistency. In this work we present results for systems of 1-centre and 2-centre Lennard-Jones mixtures at a 9/3 Lennard-Jones wall

Crystalline S-Layer Protein Monolayers Induce Water Turbulences on the Nanometer Scale

Bacterial surface layers (S-layers) have been observed as the outermost cell envelope component in a wide range of bacteria and most archaea. They are one of the most common prokaryotic cell surface structures and cover the cells completely. It is assumed that S-layers provide selection advantages to prokaryotic cells in their natural habitats since they act as protective envelopes, as structures involved in cell adhesion and surface recognition, as molecular or ion traps, and as molecular sieves in the ultrafiltration range. In order to contribute to the question of the function of S-layers for the cell, we merged high-resolution cryo-EM and small-angle X-ray scattering datasets to build a coarse-grained functional model of the S-layer protein SbpA from Lysinibacillus sphaericus ATCC 4525. We applied the Navier–Stokes and the Poisson equations for a 2D section through the pore region in the self-assembled SbpA lattice. We calculated the flow field of water, the vorticity, the electrostatic potential, and the electric field of the coarse-grained model. From calculated local changes in the flow profile, evidence is provided that both the characteristic rigidity of the S-layer and the charge distribution determine its rheological properties. The strength of turbulence and pressure near the S-layer surface in the range of 10 to 50 nm thus support our hypothesis that the S-layer, due to its highly ordered repetitive crystalline structure, not only increases the exchange rate of metabolites but is also responsible for the remarkable antifouling properties of the cell surface. In this context, studies on the structure, assembly and function of S-layer proteins are promising for various applications in nanobiotechnology, biomimetics, biomedicine, and molecular nanotechnology

Resolving domain positions of cellobiose dehydrogenase by small angle X‐ray scattering

The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron-transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain