A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.     

Correction to: In vitro cellular and proteome assays identify Wnt pathway and CDKN2A-regulated senescence affected in mesenchymal stem cells from mice after a chronic LD gamma irradiation in utero

Radiation and Environmental Biophysics -     

SAMHD1 restrains aberrant nucleotide insertions at repair junctions generated by DNA end joining

Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.     

“Organized stress” for robust scale-up of intensified production process with fed-batch seed bioreactor

Process intensification has been widely used for many years in the mammalian biomanufacturing industry to increase productivity, agility and flexibility while reducing production costs. The most commonly used intensified processes are operated using a perfusion or fed-batch seed bioreactor enabling a higher than usual seeding density in the fed-batch production bioreactor. Hence, as part of the growth phase is shifted to the seed bioreactor, there is a lower split ratio, which increases the criticality of the seed bioreactor and could impact production performance. Therefore, such intensified processes should be designed and characterized for robust process scale-up. This research work is focused on intensified processes with high seeding density inoculated from seed bioreactor in fed-batch mode. The impact of the feeding strategy and specific power input (P/V) in the seed bioreactor and on the production step with two different cell lines (CL1 and CL2) producing two different monoclonal antibodies was investigated. Cell culture performance in the production bioreactor has been improved due to more stressful conditions for the cells in the seed bioreactor and the impact of the production bioreactor P/V on the production performance was limited. This is the first reported study highlighting a positive impact of cellular stress in seed bioreactors on intensified production bioreactor with the introduction of the “organized stress” concept.     

The dynamics of ubiquitin pools within cultured human lung fibroblasts

The dynamics of ubiquitin pools within the cultured human lung fibroblast line IMR-90 were examined using solid phase immunochemical methods to quantitate free and conjugated polypeptide. Fetal calf serum was found to contain a nondialyzable factor that induced a transient accumulation of ubiquitin. During the induction, free and conjugated ubiquitin pools changed in concert so that the fraction conjugated remained constant. The induction of ubiquitin by the serum factor resulted from an enhanced rate of protein synthesis. Within experimental error no change in the first order rate constant for intracellular ubiquitin degradation was observed. Pulse-chase studies revealed ubiquitin to turn over with a half-life of 28-31 h in conditioned and freshly fed cultures. Withdrawal of serum from cultures led to a rapid decline in total ubiquitin during which the fractional level of conjugation remained constant. The accelerated ubiquitin turnover following removal of serum likely involves lysosomal autophagy since 10 mM NH4Cl led to an accumulation of the polypeptide. Since no similar effect of the lysosomotropic compound was observed in conditioned or freshly fed cultures, nonlysosomal processes are probably responsible for ubiquitin turnover under nutritional balance. The dynamics of these intracellular pools suggests that the ubiquitin ligation system is subject to regulatory constraints not previously suspected. The short half-life for ubiquitin is consistent with the apparent ability of cells to alter ubiquitin levels in response to external stimuli and stress.     

Direct analysis of α- and β-chains of hemoglobins from mammalian blood samples by nanoESI mass spectrometry during in-capillary proteolytic digestion

α- and β-chains of hemoglobins derived from several species were analyzed directly from diluted blood samples by simultaneous in-capillary proteolytic digestion and nanoESI MS and MS/MS analysis. Starting from fresh or frozen and thawed blood samples, sequence coverages of >80% were usually obtained. Only 2 h after resuspension of a dried blood spot, human origin could be demonstrated from data obtained by in-capillary tryptic digestion, nanoESI mass spectrometric analysis, and data base search. A fast and facile differentiation of closely related species by hemoglobin-derived proteolytic “marker peptides” was demonstrated for Asian (Elephas maximus) and African elephants (Loxodonta africana). Finally, amino acid sequences deduced from collision-induced dissociation experiments during in-capillary proteolytic digestion of the corresponding blood samples allowed de novo sequencing of previously unknown sequences of hemoglobin chains of the Patagonian cavy (Dolichotum patagona) and the Persian gazelle (Gazella subgutturosa subgutturosa). 100% of the α-chain sequences and more than 85% of the β-chain sequences were covered for both the species. Additionally, sequence data derived from tandem MS experiments obtained with the Q-Tof analyzer were confirmed by high resolution Fourier-transform ion cyclotron resonance mass spectrometric experiments. Accurate protein mass determination of the intact hemoglobin chains directly from the corresponding blood samples by use of a Fourier-transform ion cyclotron resonance mass spectrometer corroborated the deduced sequences of the respective α-chains. The present study demonstrates that in-capillary digestion allows fast characterization and/or sequencing of hemoglobin chains directly from blood samples.     

Biotin-Binding Proteins in the Defense of Mushrooms against Predators and Parasites

Tamavidins are fungal biotin-binding proteins (BBPs) displaying antifungal activity against phytopathogens. Here we show high toxicity of tamavidins toward nematodes, insects, and amoebae. As these organisms represent important phyla of fungal predators and parasites, we propose that BBPs are part of the chemical defense system of fungi.     

Liposome fractionation and size analysis by asymmetrical flow field-flow fractionation/multi-angle light scattering: influence of ionic strength and osmotic pressure of the carrier liquid

Asymmetrical flow field-flow fractionation (AsFlFFF)/multi-angle light scattering (MALS) was employed for studying filter-extruded liposomes in carrier solutions with different ionic strength and osmolarity. By dilution of preformed liposome suspensions with different media, only the ionic strength in the external free aqueous phase was changed. Under such conditions the liposomes were found to elute at almost identical elution times, which is in contrast to earlier studies. This may be explained by two opposing effects: (a) modulation of inter-particulate and particle-wall-repulsion effects and (b) osmotic stress-induced changes in vesicle size. The latter effect was demonstrated when analysing liposomes upon dilution in media of constant ionic strength, but varying osmotic pressure (with or without 150 mmol L^?1 sucrose supplement). The osmotic stress-induced change in liposome size was found to be size dependent. Larger liposomes appeared to both shrink and swell when exposed to hyper- or hypoosmotic media, respectively. Smaller liposomes appeared to shrink but not to swell. The potential causes of this effect are discussed.