Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris

Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions.

Bioconversion of Apple Pomace into Microbial Protein Feed Based on Extrusion Pretreatment

Apple pomace (AP) is often used directly as animal feed, while the value of feeding is limited by its low protein content. In this study, extrusion pretreatment was performed for AP, and further fermentation was carried out to improve its nutrition value. Strains suitable for extruded apple pomace (EAP) to produce high-quality microbial protein (MP) feed were screened from 12 different strains. Results showed that Aspergillus niger 3.324 (Asn), Candida utilis1314 (Cau), Geotrichum candidum 1315 (Gec), Bacillus subtilis A308 (Bas1) and Lactic acid bacteria (Lac) were screened as the dominant strains, which exhibited higher feeding value. Strongsymbiotic effect was observed in fermentation with mixed strains of Asn, Cau, Gec, and Lacat the ratio of 1:1:1:1. Compared with AP, the pure protein content in the optimized fermented EAP (FEAP) was increased by 138% accompanying with a pleasant flavor and taste. The nutrition value of FEAP was characterized by amino acid profiles, it found that FEAP was comparable to other commercial proteins with higher contents of histidine, phenylalanine, threonine and valine. Combination of fermentation and extrusion technology significantly enhanced pure protein content and nutritional composition of apple pomace, which was revalorized as a nutritive animal feed that rich in microbial protein.

Stability of the Cross Linking Component of the Biosensor Receptor Layer after Addition of Gold Nanoparticles

In this study, the stability of the receptor layer component of a biosensor after addition of gold nanoparticles was investigated. Accelerated conformational changes under the influence of Au were demonstrated. The relative percentage changes over time between the pure protein and the Au doped protein were calculated. It was shown that these changes are greater with time and exceed 20 % in the last days of the experiment.

Biophysical properties governing septin assembly

Septin filaments build structures such as rings, lattices and gauzes that serve as platforms for localizing signaling and organizing cell membranes. How cells control the geometry of septin assemblies in poorly understood. We show here that septins are isodesmic polymers, in contrast to cooperative polymerization exhibited by F-actin and microtubules. We constructed a physical model to analyze and interpret how septin assemblies change in the presence of regulators in yeast extracts. Notably filaments differ in length and curvature in yeast extract compared to pure protein indicating cellular regulators modulate intrinsic biophysical features. Combining analysis of extracts from regulatory mutants with simulations, we found increased filament flexibility and reduced filament fragmentation promote assembly of septin rings, whereas reduced flexibility in crowded environments promotes local filament alignment. This work demonstrates how tuning of intrinsic features of septin filament assembly by regulatory proteins yields a diverse array of structures observed in cells.

Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

Novel method for measuring aquatic bacterial productivity using D10-leucine based on protein synthesis rate

The most widely used method for measuring bacterial production is tritium-labeled leucine (3H-Leu). Although this method provides methodological simplicity and high sensitivity, the employment of radioactive isotopes is often restricted by regulations, particularly in field settings. In this study, we developed a non-radioactive method for measuring bacterial productivity based on the protein synthesis rate, using deuterium-labeled leucine ((CD3)2CDCD2CD(NH2)COOH; D10-Leu); the proposed method was then compared and verified with the 3H-Leu method. The procedures of the proposed method are (1) incorporation of D10-Leu by bacteria, (2) acid hydrolysis (HCl) to amino acids and (3) quantification of D10-Leu (m/z 142.10) by liquid chromatography mass spectrometry (LC-MS/MS). In the LC-MS/MS analysis, we detected a larger amount of D9-Leu (m/z 141.10) and D8-Leu (m/z 140.10) than that of D10-Leu, suggesting that incorporated D10-Leu was rapidly metabolized such as in deamination and aminotransferase reactions. The incorporation rates of D10-Leu, D10-Leu + D9-Leu (D10+D9-Leu) and D10-Leu + D9-Leu + D8-Leu (D10+D9+D8-Leu) were significantly positively correlated to that of 3H-Leu, confirming the validity of the proposed method. Since D7-Leu (m/z 139.10) could not be detected, the amount of exogenous leucine incorporated into protein can be accurately estimated through D10+D9+D8-Leu measurement. The new compound-based quantification method using stable isotope-labeled leucine can be a powerful tool to estimate pure protein synthesis rate for measuring bacterial production.

Ultrafast Backbone Protonation in Channelrhodopsin-1 Captured by Polarization Resolved Fs Vis-pump—IR-Probe Spectroscopy and Computational Methods

Channelrhodopsins (ChR) are light-gated ion-channels heavily used in optogenetics. Upon light excitation an ultrafast all-trans to 13-cis isomerization of the retinal chromophore takes place. It is still uncertain by what means this reaction leads to further protein changes and channel conductivity. Channelrhodopsin-1 in Chlamydomonas augustae exhibits a 100 fs photoisomerization and a protonated counterion complex. By polarization resolved ultrafast spectroscopy in the mid-IR we show that the initial reaction of the retinal is accompanied by changes in the protein backbone and ultrafast protonation changes at the counterion complex comprising Asp299 and Glu169. In combination with homology modelling and quantum mechanics/molecular mechanics (QM/MM) geometry optimization we assign the protonation dynamics to ultrafast deprotonation of Glu169, and transient protonation of the Glu169 backbone, followed by a proton transfer from the backbone to the carboxylate group of Asp299 on a timescale of tens of picoseconds. The second proton transfer is not related to retinal dynamics and reflects pure protein changes in the first photoproduct. We assume these protein dynamics to be the first steps in a cascade of protein-wide changes resulting in channel conductivity.

A single amino acid Gly-tag enables metal-free protein purification

Gly-tag resin precisely captures and releases a protein with one glycine at the N-terminus. The user-friendly protocol delivers analytically pure protein free of metal contaminants.