Production of Insulin in Body - Artificial Synthesis
Practical use of Recombinant DNA technology in the synthesis of human insulin requires millions of copies of the bacteria whose plasmid has been combined with the insulin gene in order to yield insulin.
Recombinant DNA technology in the synthesis of human insulin
Biological cells employ compartmentalization to overcome many difficult metabolic and physiological challenges. Eukaryotes mainly use membrane-bound organelles to sequester and control the flow of metabolites, store genetic information and segregate protein processing and export. In contrast, the majority of prokaryotes do not possess intracytoplasmic membrane systems and instead rely on protein-based approaches to achieve spatial control. However, certain specialized membrane compartments have been identified in specific bacterial strains, including polyphosphate-storing acidocalcisomes, photosynthetic thylakoids in cyanobacteria, magnetosomes in magnetotactic bacteria and pirellulosomes and anammoxosomes in morphologically complex Planctomycetes (Figure 1). Important examples for protein-based organelles include bacterial microcompartments (BMCs) like the CO2-fixing carboxysome, ferritins involved in iron homeostasis and maintaining redox balance and functionally diverse encapsulin nanocompartments (Figure 1). Encapsulating enzymes or biosynthetic pathways in semi-permeable protein organelles can increase the local concentrations of metabolites and enzymes, prevent the loss of toxic or volatile intermediates and create unique microenvironments necessary for the proper functioning of specialized enzymes. In addition, encapsulation allows for incompatible reactions and processes to take place in a single cell at the same time.
Total RNA was extracted from treated islets using Trizol reagent (Sigma, St. Louis, MO, USA), and was used as a template for cDNA synthesis with reverse transcriptase and random hexamer primers (ABI, CA, USA). Quantification of gene expression was done using SYBR Green PCR Master Mix (Eurogenetic, Belgium) using the ABI7500 fast thermal cycler. We examined expression for those genes whose primers generated a single peak in the melting curve analysis and a single, specific band in agarose gel electrophoresis. Genes analyzed in this study were NF-κB, IL1β, TNFα, NOS2a, CHOP, BCL2, CDKN1A, PDX1 and Insulin. β-actin or 18S rRNA was used as a housekeeping gene control. The primer sequences are provided in the Additional file .
Insulin production and synthesis ..
Sixty three nucleotides are required for synthesising the A chain and ninety for the B chain, plus a codon at the end of each chain,signalling the termination of protein synthesis.
Insulin production and synthesis 1
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[Synthesis and cloning of artificial zein genes]
2. Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, et al. Free fatty acid-induced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade. Diabetes. 1999 Jun;48(6):1270–4.
Chemo-enzymatic synthesis of glycosylated insulin …
In this study, we activated GPR40 using a specific small molecule agonist (CNX-011-67). Pharmacological activation of GPR40 by CNX-011-67 was very specific as an increase in cytoplasmic calcium flux was seen only in cells expressing GPR40 and no calcium flux was observed in cells which did not express GPR40. Moreover, CNX-011-67 mediated calcium flux was mediated through PLC pathway. Treatment of NIT1 cells and rat islets with CNX-011-67 showed a reduction in inflammatory signaling, inflammatory cytokines gene expression, cellular oxidative and ER stresses while increasing insulin secretion, intracellular insulin content and pro-survival signaling pathways. Taken together, GPR40 agonists provide a novel tool to counteract inflammation mediated β-cell dysfunction.