Plasmids for Optimizing Expression of Recombinant Proteins in <em>E. coli</em>

Plasmids are important vectors for the transfer of genetic material among microbes. The transfer of plasmids causes transmission of genes involved in pathogenesis and survival, to the host bacteria leading to their evolution and adaptation to diverse environmental conditions. A large number of plasmids of varying sizes have been discovered and isolated from various microorganisms. Plasmids are also valuable tools to genetically manipulate microbes for various purposes including production of recombinant proteins. Escherichia coli is the most preferred microbe for production of recombinant proteins, due to rapid growth rate, cost-effectiveness, high yield of the recombinant proteins and easy scale-up process. Several plasmids have been designed to optimize the expression of heterologous proteins in E. coli. In order to circumvent the issues of protein refolding, the codon usage in E. coli, the absence of post-translational modifications, such as glycosylation and low recovery of functionally active recombinant proteins, various plasmids have been designed and constructed. This chapter summarizes the recent technological advancements that have extended the use of the E. coli expression system to produce more complex proteins, including glycosylated recombinant proteins and therapeutic antibodies

Comparative molecular studies of halophilic bacteria from saline water and soil in the Saudi environment

Halophilic bacteria are a microorganism that grows optimally in the presence of the very high concentration of sodium chloride. Halophiles are vital sources of various enzymes including hydrolases, which are very stable and catalytically highly efficient at high salt concentration and other extreme conditions such as high temperature, pH and presence of organic solvents.  Several hydrolases such as amylases, proteases, and lipases have been obtained from halophilic bacteria and are commonly used for various industrial applications. We initiated a screening project to isolate and characterize the halophilic bacteria from the Red Sea, which is one of the saltiest bodies of water in the world. Water and soil samples, collected from the Red Sea coast, Jeddah, Saudi Arabia, were screened for isolation of halophilic bacteria. Ten bacterial isolates were obtained, which were characterized by biochemical tests and 16S rRNA gene sequencing. Hydrolase producing bacteria among the isolates were screened by plate assay on starch and gelatin agar plates for amylase and protease, respectively.  Two bacterial isolates i.e Bacillus haynesii and Enterobacter cloacae subsp. were found to possess significant amylase and protease activity. Further characterization of both the strains is in progress

Effect of gibberellins and ascorbic acid treatment on phytic acid and micronutrients dialyzability in germinated biofortified wheat seeds

Introduction: Phytic acid chelate minerals, including Fe and Zn and render them inaccessible once ingested by human beings. The evaluation of differences in the dialyzability of macronutrients including Fe and Zn in various wheat derivatives is therefore important for the enhancement of nutritional quality of grains. Objectives: The objective of current study was to improve the micronutrient content in wheat grain. Methods: During germination (12, 24, 48, 72, 96 h), effects of gibberellins and ascorbic acid on phytic acid content as well as dialyzability of iron and zinc of wheat derivatives were determined. Results: The phytic acid content in wheat flour was determined and it was found 7.61 to 7.48 mg/g. After the treatment with gibberellins, it was significantly reduced from 8.68 to 21.6 % and 9.65 to 20.9 % with ascorbic acid. In wheat flour dialyzabilty of Fe was 4.53 to 8.97 mg/kg. After germination, Fe content was increased from 9.77 to 32.0 % with gibberellin, and 13.9 to 31.0 % with ascorbic acid. Moreover, with gibberellins, Zn content was increased from 8.68 to 21.6 % and 9.65 to 20.9 % with ascorbic acid, respectively. Conclusion: These results suggested that gibberellins as well as ascorbic acid can be exploited to improve the dialyzability of iron and zinc content due to reduced antinutrient i. e phytic acid and make the minerals available for the absorption in monogastric animals including human beings

Anti-bacterial activity of Ricinus communis L. against bacterial pathogens Escherichia coli and Klebsiella oxytoca as evaluated by Transmission electron microscopy

The emergence of multidrug-resistant (MDR) microbes has become one of the major threat globally. Infectious diseases are the second leading cause of death, two-third of which are caused by Gram-negative bacteria. The increasing number of multidrug resistant (MDR) microbes is quite alarming and has raised the necessity of development of new antibacterial drugs. Escherichia coli and Klebsiella have been reported among the top most resistance-developing pathogens. Ricinus communis is an important medicinal plant reported to possess antimicrobial phytochemicals such as α-pinene. The hexane treated crude ethanolic extract of R. communis was evaluated against Gram-negative bacteria E. coli and Klebsiella oxytoca. The agar well diffusion assay was used to determine the antibacterial activity. In the present study, we have shown experimentally that leaf extract of R. communis can induce the deterioration of the inner and outer cell membranes of E. coli and K. oxytoca and decrease their viability at a concentration of 50 mg/ml. Transmission electron microscopic results revealed cell membrane damage, cellular disintegration and release of cytoplasmic content, leading to cell death. To our knowledge, this is the first study of the antibacterial activity of R. communis against E. coli and K. oxytoca by Transmission electron microscopy. The ultramicroscopic observations showed that the phytochemical present in the leaf extract of R. communis could penetrate the bacterial cell, causing rupture of cell membranes and hence confirm the cytotoxic and antimicrobial property of R. communis

Therapeutic role of Ricinus communis L. and its bioactive compounds in disease prevention and treatment

Ricinus communis L. (R. communis), commonly known as castor oil plant, is used as a traditional natural remedy or folkloric herb for the control and treatment of a wide range of diseases around the globe. Various studies have revealed the presence of diverse phytochemicals such as alkaloids, flavonoids, terpenes, saponins, phenolic compounds such as kaempferol, gallic acid, ricin, rutin, lupeol, ricinoleic acid, pinene, thujone and gentisic acid. These phytochemicals have been responsible for pharmacological and therapeutic effects, including anticancer, antimicrobial, insecticidal, antioxidant, anti-diabetic, antinociceptive, anti-inflammatory, bone regenerative, analgesic, and anticonvulsant activity. R. communis harbours phytochemicals which have been shown to target peroxisome proliferator activated receptor (PPAR), nuclear factor NF- κ -B, cytochrome p450, P38 mitogen-activated protein kinases kinase (p38 MAPK), tumor protein P53, B-cell lymphoma-extra-large (Bcl-xL) and vascular endothelial growth factor receptor-2 (VEGFR-2). Considering its wide variety of phytochemicals, its pharmacological activity and the subsequent clinical trials, R. communis could be a good candidate for discovering novel complementary drugs. Further experimental and advanced clinical studies are required to explore the pharmaceutical, beneficial therapeutic and safety prospects of R. communis with its phytochemicals as a herbal and complementary medicine for combating various diseases and disorders

Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum

Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 107 cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (Km=1.7 &#x03BC;M). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target

Gene expression profiling to elucidate the pharmacological and toxicological effects of Ricinus communis L. leaf extract in mammalian cells

Ricinus communis is a traditional medicinal plant which has been utilized for centuries for treatment of various conditions. Due to the presence of diverse phytochemicals, Ricinus is an outstanding natural resource to discover new drugs for various diseases such as diabetes, cancer, arthritis, ulcer and asthma. In this study, we performed whole-genome gene expression profiling using RNA-Seq to determine the differentially expressed genes in a mammalian cell line after exposure to Ricinus leaf extract and elucidate their pharmacological effects in order to support its ethnomedicinal uses. Various genes involved in cancer, inflammation, atherosclerosis and diabetes were found to be differentially regulated after exposure to sub-lethal concentrations of the Ricinus extract in MCF7 cells. An important gene involved in cancer progression and metastasis, that is, PIK3R3 (Phosphatidylinositol 3-kinase regulatory subunit gamma), was downregulated in MCF7 cells after treatment with Ricinus extract. PIK3R3 is an important component of the PI3K/AKT signalling pathway which is essential for cell proliferation, angiogenesis, inhibition of apoptosis and metastasis to distant organs. The Ricinus extract downregulated the expression of DPP4 (Dipeptidyl peptidase-4) and upregulated the expression of PPAR-γ (Peroxisome proliferator-activated receptor gamma) which are crucial in controlling blood glucose levels. Expression of TNFAIP6 (Tumor necrosis factor-inducible gene 6), which is shown to mediate anti-inflammatory and protective effects, was increased after treatment with Ricinus extract. We also analyzed the genes which might also confer toxicity. Our gene expression profiling data corroborate the potential therapeutic benefits of Ricinus communis plant