Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different ...purification tags have been developed, including short peptides, epitopes, folded protein domains, non-chromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research.
Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism ...by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3' single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.
The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags ...and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification.
DNA polymerase theta (Pol θ)-mediated end joining (TMEJ) has been implicated in the repair of chromosome breaks, but its cellular mechanism and role relative to canonical repair pathways are poorly ...understood. We show that it accounts for most repairs associated with microhomologies and is made efficient by coupling a microhomology search to removal of non-homologous tails and microhomology-primed synthesis across broken ends. In contrast to non-homologous end joining (NHEJ), TMEJ efficiently repairs end structures expected after aborted homology-directed repair (5′ to 3′ resected ends) or replication fork collapse. It typically does not compete with canonical repair pathways but, in NHEJ-deficient cells, is engaged more frequently and protects against translocation. Cell viability is also severely impaired upon combined deficiency in Pol θ and a factor that antagonizes end resection (Ku or 53BP1). TMEJ thus helps to sustain cell viability and genome stability by rescuing chromosome break repair when resection is misregulated or NHEJ is compromised.
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•Pol θ directs end joining by coupling a microhomology search to synthesis across ends•Pol θ-mediated end joining is critical when either canonical repair pathway fails•Pol θ is also essential when cells aberrantly regulate resection of chromosome breaks•Pol θ-dependent repair can both suppress and promote genome instability
Wyatt et al. show that Polymerase θ accounts for most microhomology-dependent chromosome break repair and outline a mechanism that makes this pathway efficient and flexible. They define biologically relevant substrates, determine that it is essential in cells that inappropriately regulate resection, and show that it can both positively and negatively affect genome stability.
Recent outbreaks of animal-borne emerging infectious diseases have likely been precipitated by a complex interplay of changing ecological, epidemiological and socio-economic factors. Here, we develop ...modelling methods that capture elements of each of these factors, to predict the risk of Ebola virus disease (EVD) across time and space. Our modelling results match previously-observed outbreak patterns with high accuracy, and suggest further outbreaks could occur across most of West and Central Africa. Trends in the underlying drivers of EVD risk suggest a 1.75 to 3.2-fold increase in the endemic rate of animal-human viral spill-overs in Africa by 2070, given current modes of healthcare intervention. Future global change scenarios with higher human population growth and lower rates of socio-economic development yield a fourfold higher likelihood of epidemics occurring as a result of spill-over events. Our modelling framework can be used to target interventions designed to reduce epidemic risk for many zoonotic diseases.
T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with ...a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.
We draw from cognitive science literature on rule‐based thinking to develop and empirically test a theoretical framework of entrepreneurial opportunity evaluation. We argue that entrepreneurs make ...use of socially constructed rules to discern the attractiveness of an opportunity, for them, specifically. Using conjoint analysis data of 498 decisions made by 62 entrepreneurs, we find that entrepreneurs' use of rules regarding opportunity novelty, resource efficiency, and worst‐case scenario significantly influences entrepreneurs' evaluations of opportunities and that individual differences in opportunity market and technology knowledge augment the effect of the rules on opportunity attractiveness. Additionally, we document that the worst‐case scenario diminishes the positive effect of other rule criteria (e.g. novelty, resource efficiency) on opportunity evaluation and that market and technology knowledge further influence the negative effects of the worst‐case scenario.
This volume of articles in English by an international team of scholars presents new critical perspectives on the first principles of J.G. Fichte's Wissenschaftslehre and some of the key ...sub-disciplines of his philosophy.
Ecological theory suggests that habitat disturbance differentially influences distributions of habitat generalist and specialist species. While well-established for macroorganisms, this theory has ...rarely been explored for microorganisms. Here we tested these principles in permeable (sandy) sediments, ecosystems with much spatiotemporal variation in resource availability and physicochemical conditions. Microbial community composition and function were profiled in intertidal and subtidal sediments using 16S rRNA gene amplicon sequencing and metagenomics, yielding 135 metagenome-assembled genomes. Community composition and metabolic traits modestly varied with sediment depth and sampling date. Several taxa were highly abundant and prevalent in all samples, including within the orders Woeseiales and Flavobacteriales, and classified as habitat generalists; genome reconstructions indicate these taxa are highly metabolically flexible facultative anaerobes and adapt to resource variability by using different electron donors and acceptors. In contrast, obligately anaerobic taxa such as sulfate reducers and candidate lineage MBNT15 were less abundant overall and only thrived in more stable deeper sediments. We substantiated these findings by measuring three metabolic processes in these sediments; whereas the habitat generalist-associated processes of sulfide oxidation and fermentation occurred rapidly at all depths, the specialist-associated process of sulfate reduction was restricted to deeper sediments. A manipulative experiment also confirmed habitat generalists outcompete specialist taxa during simulated habitat disturbance. Together, these findings show metabolically flexible habitat generalists become dominant in highly dynamic environments, whereas metabolically constrained specialists are restricted to narrower niches. Thus, an ecological theory describing distribution patterns for macroorganisms likely extends to microorganisms. Such findings have broad ecological and biogeochemical ramifications.
A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag ...self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting as an initial affinity tag. Combining the heparin-binding tag (HB-tag) with the self-removing iCapTag™ provides a means for removing both tags in a subsequent self-cleaving step. The result is a convenient platform for the purification of diverse tagless proteins with a range of isoelectric points and molecular weights. In this work, we demonstrate a dual column process in which the tagged protein of interest is first captured from an E. coli cell lysate using a cation exchange column via a fused heparin-binding affinity tag. The partially purified protein is then diluted and loaded onto an iCapTag™ split-intein column, washed, and then incubated overnight to release the tagless target protein from the bound tag. Case studies are provided for enhanced green fluorescent protein (eGFP), beta galactosidase (βgal), maltose binding protein (MBP) and beta lactamase (βlac), where overall purity and host cell DNA clearance is provided. Overall, the proposed dual column process is shown to be a scalable platform technology capable of accessing both the high dynamic binding capacity of ion exchange resins and the high selectivity of affinity tags for the purification of recombinant proteins.
•Target proteins were expressed in fusion with the highly charged HB-tag and the self-removing iCapTag™.•The resulting platform process required mild pH conditions and resulted in highly pure tagless bioactive products.•The process was designed to be efficient with multiple buffers being used for different purposes.
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