The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli : a comparison with the traditional gene fusion technology

The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.The financial support of the EMBL Heidelberg, Germany and Fundacao para a Ciencia e Tecnologia (FCT), Portugal, is acknowledged: the fellowship SFRH/BD/46482/2008 to Sofia J. Costa and the project PTDC/CVT/103081/2008. The authors wish to acknowledge Anne-Claude Gavin for providing four of the constructs for this study (RVS167, SPO14, YPK1, and YPK2) and Emmanuel Poilpre for the experimental help (both from the EMBL Heidelberg, Germany)

An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family

A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol zeta (a family-B translesion synthesis polymerase). Inactivation of the 3'–5' proofreading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with amarked tendency tomake changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an errorprone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA

Erst kommt das Fressen: Kerstin Jacobsson und Jonas Lindblom betrachten den Tierschutzaktivismus aus moralsoziologischer Perspektive

Kerstin Jacobsson / Jonas Lindblom: Animal Rights Activism: A Moral-Sociological Perspective on Social Movements. Amsterdam: Amsterdam University Press 2017. 978-908964764

Vom Mitgefühl zur Fairness: Michael Tomasello schreibt eine Naturgeschichte der menschlichen Moral

Michael Tomasello: Eine Naturgeschichte der menschlichen Moral. Frankfurt am Main: Suhrkamp Verlag 2016. 978-3-518-58695-

Hoffnungsvoll in eine ungewisse Zukunft: Rezension zu "Radikale Hoffnung: Ethik im Angesicht kultureller Zerstörung" von Jonathan Lear

Jonathan Lear: Radikale Hoffnung: Ethik im Angesicht kultureller Zerstörung. Berlin: Suhrkamp 2021. 978-3-518-58759-

Die Macht der Stimmungen: Heinz Bude über Stimmungen in Deutschland und Stimmung als soziologische Schlüsselkategorie

Heinz Bude: Das Gefühl der Welt: Über die Macht von Stimmungen. München: Hanser 2016. 978344625065

Das höchste Gut: Didier Fassin versucht sich an einer Analyse der Ungleichheit menschlicher Leben

Didier Fassin: Das Leben: Eine kritische Gebrauchsanweisung. Berlin: Suhrkamp 2017. 978-3-518-58710-

Hypothesis-generating analysis of the impact of non-damaging metabolic acidosis on the transcriptome of different cell types : integrated stress response (ISR) modulation as general transcriptomic reaction to non-respiratory acidic stress?

Extracellular pH is an important parameter influencing cell function and fate. Microenvironmental acidosis accompanies different pathological situations, including inflammation, hypoxia and ischemia. Research focussed mainly on acidification of the tumour micromilieu and the possible consequences on proliferation, migration and drug resistance. Much less is known regarding the impact of microenvironmental acidosis on the transcriptome of non-tumour cells, which are exposed to local acidosis during inflammation, hypoxia, ischemia or metabolic derailment. In the present hypothesis-generating study, we investigated the transcriptional impact of extracellular acidosis on five non-tumour cell types of human and rat origin, combining RNA-Sequencing and extensive bioinformatics analyses. For this purpose, cell type-dependent acidosis resiliences and acidosis-induced transcriptional changes within these resilience ranges were determined, using 56 biological samples. The RNA-Sequencing results were used for dual differential-expression analysis (DESeq and edgeR) and, after appropriate homology mapping, Gene Ontology enrichment analysis (g:Profiler), Ingenuity Pathway Analysis (IPA®), as well as functional enrichment analysis for predicted upstream regulators, were performed. Extracellular acidosis led to substantial, yet different, quantitative transcriptional alterations in all five cell types. Our results identify the regulator of the transcriptional activity NCOA5 as the only general acidosis-responsive gene. Although we observed a species- and cell type-dominated response regarding gene expression regulation, Gene Ontology enrichment analysis and upstream regulator analysis predicted a general acidosis response pattern. Indeed, they suggested the regulation of four general acidosis-responsive cellular networks, which comprised the integrated stress response (ISR), TGF-β signalling, NFE2L2 and TP53. Future studies will have to extend the results of our bioinformatics analyses to cell biological and cell physiological validation experiments, in order to test the refined working hypothesis here