Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System.

Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.

Co-circulation of three camel coronavirus species and recombination of MERS-CoVs in Saudi Arabia.

Outbreaks of Middle East respiratory syndrome (MERS) raise questions about the prevalence and evolution of the MERS coronavirus (CoV) in its animal reservoir. Our surveillance in Saudi Arabia in 2014 and 2015 showed that viruses of the MERS-CoV species and a human CoV 229E-related lineage co-circulated at high prevalence, with frequent co-infections in the upper respiratory tract of dromedary camels. viruses of the betacoronavirus 1 species, we found that dromedary camels share three CoV species with humans. Several MERS-CoV lineages were present in camels, including a recombinant lineage that has been dominant since December 2014 and that subsequently led to the human outbreaks in 2015. Camels therefore serve as an important reservoir for the maintenance and diversification of the MERS-CoVs and are the source of human infections with this virus.

Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives.

Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, cost-effectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.

Role of toxicogenomics in the development of safe, efficacious and novel anti-microbial therapies.

Over the last two decades, occurrence of bacterial resistance to commonly used antibiotics has necessitated the development of safer and more potent anti-microbial drugs. However, the development of novel antibiotics is severely hampered by adverse side effects, such as drug-induced liver toxicity. Several antibacterial drugs are known to have the potential to cause severe liver damage. The major challenge in developing novel anti-microbial drugs is to predict, with certain amount of probability, the drug-induced toxicity during the pre-clinical stages, thus optimizing and reducing the time and cost of drug development. Toxicogenomics approach is generally used to harness the potential of genomic tools and to understand the physiological basis of drug-induced toxicity based on the in-depth analysis of Metagenomic data sets, i.e., transcriptional, translational or metabolomic profiles. Toxicogenomics, therefore, represents a new paradigm in the drug development process, and is anticipated to play an invaluable role in future to develop safe and efficacious medicines, by predicting the toxic potential of a new chemical entity (NCE) in early stages of drug discovery. This review examines the toxicogenomic approach in predicting the safety/toxicity of novel anti-microbial drugs, and analyses the promises, pitfalls and challenges of applying this powerful technology to the drug development process.