Production of native recombinant proteins using a novel split intein affinity technology.

Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a CC-intein tag is engineered into a protein of interest for binding to a NC-intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the NC-intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10-20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the CC-intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems.

Identification, tracking, and sizing of nano-sized particles using dual-view polarization-resolved digital holography and T-matrix modeling.

In this paper, we demonstrate how polarization-resolved holography can be used to determine if a particle is spherical or not and to estimate the size information of nanoparticles. The T-matrix method is used to model the scattered light from both spheres and spheroids. A dual-view polarization-resolved imaging system is used in order to obtain polarization ratio angles (ß1,ß2). From the obtained ß1 and ß2, it is possible to estimate whether or not a particle is spherical. It is found that nonsphericity only has a minor effect up to around sizes of 120 nm, and for that range, a spherical approximation is valid. For larger particles, the orientation influences the polarization response greatly. The size of a nonspherical particle can be estimated from the polarization ratio angles. The upper limit we can estimate unambiguously is around 200 nm. Finally, the model is applied to experimental measurements of naturally occurring particles in purified water. From the measurements, it is possible to separate spherical from nonspherical particles and also give a rough estimate of the size.

Polarization-resolved dual-view holographic system for 3D inspection of scattering particles.

A novel dual-view polarization-resolved pulsed holographic system for particle measurements is presented. Both dual-view configuration and polarization-resolved registration are well suited for particle holography. Dual-view registration improves the accuracy in the detection of 3D position and velocities, and polarization-resolved registration provides polarization information about individual particles. The necessary calibrations are presented, and aberrations are compensated for by mapping the positions in the two views to positions in a global coordinate system. The system is demonstrated on a sample consisting of 7 µm spherical polystyrene particles dissolved in water in a cuvette. The system is tested with different polarizations of the illumination. It is found that the dual view improves the accuracy significantly in particle tracking. It is also found that by having polarization-resolved holograms, it is possible to separate naturally occurring sub-micrometer particles from the larger, 7 µm seeding particles.

Improved particle position accuracy from off-axis holograms using a Chebyshev model.

Side scattered light from micrometer-sized particles is recorded using an off-axis digital holographic setup. From holograms, a volume is reconstructed with information about both intensity and phase. Finding particle positions is non-trivial, since poor axial resolution elongates particles in the reconstruction. To overcome this problem, the reconstructed wavefront around a particle is used to find the axial position. The method is based on the change in the sign of the curvature around the true particle position plane. The wavefront curvature is directly linked to the phase response in the reconstruction. In this paper we propose a new method of estimating the curvature based on a parametric model. The model is based on Chebyshev polynomials and is fit to the phase anomaly and compared to a plane wave in the reconstructed volume. From the model coefficients, it is possible to find particle locations. Simulated results show increased performance in the presence of noise, compared to the use of finite difference methods. The standard deviation is decreased from 3-39 µm to 6-10 µm for varying noise levels. Experimental results show a corresponding improvement where the standard deviation is decreased from 18 µm to 13 µm.

Off-axis digital holographic particle positioning based on polarization-sensitive wavefront curvature estimation.

Poor axial resolution in holographic particle imaging applications makes particle positioning in 3D space more complex since the positions are not directly obtained. In this paper we estimate the axial position of micrometer particles by finding the location where the wavefront curvature from the scattered light becomes zero. By recording scattered light at 90° using off-axis holography, the complex amplitude of the light is obtained. By reconstruction of the imaged scene, a complex valued volume is produced. From this volume, phase gradients are calculated for each particle and used to estimate the wavefront curvature. From simulations it is found that the wavefront curvature became zero at the true axial position of the particle. We applied this metric to track an axial translation experimentally using a telecentric off-axis holographic imaging system with a lateral magnification of M=1.33. A silicon cube with molded particles inside was used as sample. Holographic recordings are performed both before and after a 100 µm axial translation. From the estimated positions, it was found that the mean displacement of particles between recordings was 105.0 µm with a standard deviation of 25.3 µm.

Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting.

We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant, cancer relevant tyrosine phosphorylated (pTyr) proteins. The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns. The same anti-phosphotyrosine specific antibody, 4G10, was used for both approaches. The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib. This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels. Enrichment of pTyr proteins was found highly advantageous for the outcome. Out of a simplified 2-D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins, differential analysis as well as protein identification by mass spectrometry (MS) was possible.

Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells.

Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inflammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inflammation. To facilitate characterization and development of specific and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione S-transferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was purified in a single step by glutathione-affinity chromatography followed by site-specific cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-affinity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.

A proteomics approach to the study of absorption, distribution, metabolism, excretion, and toxicity.

A proteomics approach was used to identify liver proteins that displayed altered levels in mice following treatment with a candidate drug. Samples from livers of mice treated with candidate drug or untreated were prepared, quantified, labeled with CyDye DIGE Fluors, and subjected to two-dimensional electrophoresis. Following scanning and imaging of gels from three different isoelectric focusing intervals (3-10, 7-11, 6.2-7.5), automated spot handling was performed on a large number of gel spots including those found to differ more than 20% between the treated and untreated condition. Subsequently, differentially regulated proteins were subjected to a three-step approach of mass spectrometry using (a) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting, (b) post-source decay utilizing chemically assisted fragmentation, and (c) liquid chromatography-tandem mass spectrometry. Using this approach we have so far resolved 121 differentially regulated proteins following treatment of mice with the candidate drug and identified 110 of these using mass spectrometry. Such data can potentially give improved molecular insight into the metabolism of drugs as well as the proteins involved in potential toxicity following the treatment. The differentially regulated proteins could be used as targets for metabolic studies or as markers for toxicity.