C–H insertion as a key step to spiro-oxetanes, scaffolds for drug discovery

A new route to spiro-oxetanes, potential scaffolds for drug discovery, is described. The route is based on the selective 1,4-C–H insertion reactions of metallocarbenes, generated from simple carbonyl precursors in flow or batch mode, to give spiro-β-lactones that are rapidly converted into spiro-oxetanes. The three-dimensional and lead like-properties of spiro-oxetanes is illustrated by the conversion of the 1-oxa-7-azaspiro[3,5]nonane scaffold into a range of functionalized derivatives

Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

<p>Abstract</p> <p>Background</p> <p>The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.</p> <p>Results</p> <p>A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in <it>P. pastoris</it>, was cloned in frame with the <it>Saccharomyces cerevisiae </it>α-factor secretory signal and integrated into the genome of <it>P. pastoris </it>strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L^-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth.</p> <p>Conclusions</p> <p>A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using <it>Pichia </it>based expression systems, thus significantly increasing the efficiency of insulin manufacture.</p

TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing

TDP-43 is a highly conserved nuclear factor of yet unknown function that binds to ug-repeated sequences and is responsible for cystic fibrosis transmembrane conductance regulator exon 9 splicing inhibition. We have analyzed TDP-43 interactions with other splicing factors and identified the critical regions for the protein/protein recognition events that determine this biological function. We show here that the C-terminal region of TDP-43 is capable of binding directly to several proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with well known splicing inhibitory activity, in particular, hnRNP A2/B1 and hnRNP A1. Mutational analysis showed that TDP-43 proteins lacking the C-terminal region could not inhibit splicing probably because they were unable to form the hnRNP-rich complex involved in splicing inhibition. Finally, through splicing complex analysis, we show that splicing inhibition mediated by TDP-43 occurs at the earliest stages of spliceosomal assembly

Improving human interferon-\u3b2 production in mammalian cell lines by insertion of an intronic sequence within its naturally uninterrupted gene

Human \u3b2-interferon is used extensively as a therapeuticagent in a wide variety of diseases, ranging from multi-ple sclerosis to viral infections. At present, the mostcommon source of interferon-\u3b2 is derived from CHO(Chinese-hamster ovary) cells. Interestingly, however,the IFNB gene is characterized by a lack of intronicsequences and therefore does not undergo splicingduring its expression pathway. As nuclear processing ofpre-mRNA molecules has often been demonstrated toimprove production yields of recombinant molecules,we have inserted a heterologous intronic sequence atdifferent positions within the IFNB gene and analysedits effects on protein production. The results obtainedin the present study show that the position of introninsertion has profound effects on the expressionlevels of the IFNB gene and on the nuclear/cytoplasmdistribution levels of its mRNA as determined by FISH(fluorescent in situ hybridization) analysis of stablytransfected clones. In conclusion, our results provideadditional evidence that insertion of intronic sequencesmay be used to improve protein expression efficiencyalso in molecules that do not normally undergo anysplicing process

Overview of insulin production and purification process from <i>Pichia pastoris</i> fermentation culture.

<p>Overview of insulin production and purification process from <i>Pichia pastoris</i> fermentation culture.</p