Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli

U ovom radu predstavljena je ekspresija rekombinantne atipične aspartatne proteinaze heljde (Fagopyrum esculentum) bogate cisteinom, gde su testirana različita ekspresiona svojstva pet sojeva E. coli. Takođe je analiziran i uticaj fuzionih partnera (His6 i MBP) na efikasnost ekspresije. U slučaju His6-FeAPL1, dobijena je velika količina nerastvornog proteina, smeštenog u inkluzionim telima. S druge strane, MBP-FeAPL1 je bio lokalizovan i u citoplazmi i u inkluzionim telima u oba upotrebljena soja E. coli (BL21 i Rosetta-gami). Međutim, samo za rekombinantni protein proizveden u soju Rosetta-gami, dokazana je proteolitička aktivnost na supstratu BSA, pri pH 3,0. Rezultati su takođe ukazali da FeAPL1 sadrži PRO segment, čije je odstranjivanje neophodno za njegovu proteolitičku aktivnost. Aktivnost FeAPL1, pokazana samo u soju Rosetta-gami, gde je moguće formiranje disulfidnih veza, ukazuje na značaj 12 cisteina u uspostavljanju pravilne strukture koja omogućava funkcionalnost enzima.Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His6-FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation, indicated the importance of the twelve cysteine residues for correct folding and functionality

Replication-Independent Histone Variant H3.3 Controls Animal Lifespan through the Regulation of Pro-longevity Transcriptional Programs

Chromatin structure orchestrates the accessibility to the genetic material. Replication-independent histone variants control transcriptional plasticity in postmitotic cells. The life-long accumulation of these histones has been described, yet the implications on organismal aging remain elusive. Here, we study the importance of the histone variant H3.3 in Caenorhabditis elegans longevity pathways. We show that H3.3-deficient nematodes have negligible lifespan differences compared to wild-type animals. However, H3.3 is essential for the lifespan extension of C. elegans mutants in which pronounced transcriptional changes control longevity programs. Notably, H3.3 loss critically affects the expression of a very large number of genes in long-lived nematodes, resulting in transcriptional profiles similar to wild-type animals. We conclude that H3.3 positively contributes to diverse lifespan-extending signaling pathways, with potential implications on age-related processes in multicellular organisms

A crucial role of Mim2 in the biogenesis of mitochondrial outer membrane proteins

Most of the mitochondrial outer membrane (MOM) proteins contain helical transmembrane domains. Some of the single span proteins and all known multiple span proteins are inserted into the membrane in a pathway which depends on the MOM protein Mitochondrial Import 1 (Mim1). So far it has been unknown whether additional proteins are required for this process. Here we describe the identification and characterization of Mim2, a novel protein of the mitochondrial outer membrane that has a crucial role in the biogenesis of MOM helical proteins. Mim2 physically and genetically interacts with Mim1 and both proteins form the MIM complex. Cells lacking Mim2 exhibit a severely reduced growth rate and lower steady state levels of helical MOM proteins. In addition, absence of Mim2 leads to compromised assembly of the translocase of the outer mitochondrial membrane (TOM complex), hampered mitochondrial protein import, and defects in mitochondrial morphology. In summary, the current study demonstrates that Mim2 is a novel central player in the biogenesis of MOM proteins.</jats:p