Heterologous expression and enhanced production of ß-1, 4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.

Long-Term Assessment of AAV-Mediated Zinc Finger Nuclease Expression in the Mouse Brain.

Gene editing tools like TALENs, ZFNs and Crispr/Cas now offer unprecedented opportunities for targeted genetic manipulations in virtually all species. Most of the recent research in this area has concentrated on manipulation of the genome in isolated cells, which then give rise to transgenic animals or modified stem cell lines. Much less is known about applicability of genetic scissors in terminally differentiated, non-dividing cells like neurons of the adult brain. We addressed this question by expression of a pair of ZFNs targeting the murine cathepsin D gene in CNS neurons by means of an optimized AAV viral vector. We show that ZFN expression resulted in substantial depletion of cathepsin D from neuronal lysosomes, demonstrating a robust gene deletion. Importantly, long-term ZFN expression in CNS neurons did not impair essential neuronal functionality and did not cause inflammation or neurodegeneration, suggesting that potent genetic scissors can be expressed safely in the mouse brain. This finding opens up new venues to create novel research models for neurodegenerative disorders.

Dermatomycoses: challenges and human immune responses.

The most prevalent skin infections are mainly caused by species of dermatophytes of the genera Trichophyton, Microsporum, and Epidermophyton that infect keratinized tissues and stratum corneum of skin and hair. Besides proteases with putative role of kinases and other enzymes, immune modulators are abundantly secreted during infection as well. The molecular mechanism used by the dermatophytes to infect and counteract the host immune response is not well understood. The defense against infections basically depends on the host's immune responses to metabolites of the fungi, virulence of the infecting strain or species and anatomical site of the infection. The two aspects of the immune system, the immediate hypersensitivity and delayed-type hypersensitivity against dermatophytes may be crucial to the progression and severity of skin infection. Management of the infection through species identification and molecular diagnostic techniques as well as use of novel targeted drugs in addition to conventional anti-fungal compounds is of great importance in dealing with disease onsets and outbreaks. Here we reviewed the fungal skin infections elucidating their biologic and immunologic characteristics. Reaction to fungal invasion by the infected epithelial tissue on the host side is also discussed. Moreover, determinants of protective immunity and treatment options are focused that could confer long-lasting resistance to infection.

Plant-bacterium interactions analyzed by proteomics.

The evolution of the plant immune response has resulted in a highly effective defense system that is able to resist potential attack by microbial pathogens. The primary immune response is referred to as pathogen associated molecular pattern (PAMP) triggered immunity and has evolved to recognize common features of microbial pathogens. In response to the delivery of pathogen effector proteins, plants acquired R proteins to fight against pathogen attack. R-dependent defense response is important in understanding the biochemical and cellular mechanisms and underlying these interactions will enable molecular and transgenic approaches for crops with increased biotic resistance. Proteomic analyses are particularly useful for understanding the mechanisms of host plant against the pathogen attack. Recent advances in the field of proteome analyses have initiated a new research area, i.e., the analysis of more complex microbial communities and their interaction with plant. Such areas hold great potential to elucidate, not only the interactions between bacteria and their host plants, but also of bacteria-bacteria interactions between different bacterial taxa, symbiotic, pathogenic bacteria, and commensal bacteria. During biotic stress, plant hormonal signaling pathways prioritizes defense over other cellular functions. Some plant pathogens take advantage of hormone dependent regulatory system by mimicking hormones that interfere with host immune responses to promote virulence (vir). In this review, it is discussed the cross talk that plays important role in response to pathogens attack with different infection strategies using proteomic approaches.

Identification of novel autoantigen in the synovial fluid of rheumatoid arthritis patients using an immunoproteomics approach.

Rheumatoid arthritis (RA) is a chronic, autoimmune and inflammatory joint disease with a poorly understood etiology. Despite widespread diagnostic use of anti-citrullinated protein antibodies and rheumatoid factor proteins there is a strong demand for novel serological biomarkers to improve the diagnosis this disease. The present study was aimed to identify novel autoantigens involved in rheumatoid arthritis (RA) pathogenesis through immune-proteomic strategy. Synovial fluid samples from clinically diagnosed RA patients were separated on two-dimensional gel electrophoresis (2-DE). Samples from patients with non-RA rheumatisms (osteoarthritis and trauma) were used as controls. Immunoreactive proteins were spotted by Western blotting followed by identification through Q-TOF mass spectrometer analysis. Forty Western blots were generated using plasma from ten individual RA patients and 33 reactive spots were identified, 20 from the high molecular weight (HMW) gel and 13 from the low molecular weight (LMW) gel. Among the 33 common immunogenic spots, 18 distinct autoantigens were identified, out of which 14 are novel proteins in this context. Expression analysis of five important proteins, vimentin, gelsolin, alpha 2 HS glycoprotein (AHSG), glial fibrillary acidic protein (GFAP), and α1B-glycoprotein (A1BG) by Western blot analysis using their specific antibodies revealed their higher expression in RA synovial fluid as compared to non-RA samples. Recombinantly expressed GFAP and A1BG protein were used to develop an in-house ELISA to quantify the amount of autoantibodies in the RA patients. RA patients revealed an increase in the expression of GFAP and A1BG in the plasma as compared to osteoarthritis patients. Therefore, GFAP and A1BG can be proposed as potential new autoantigens of diagnostic importance for RA subjects. Further characterization of these proteins in rheumatoid arthritis will be helpful in understanding the role of these proteins in the disease pathogenesis providing new diagnostic tool with better specificity and accurate detection of the disease.

Gossypium arboreum GHSP26 enhances drought tolerance in Gossypium hirsutum.

Heat-shock proteins (HSP) are molecular chaperones for protein molecules. These proteins play an important role in protein-protein interactions such as, folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. A small HSP gene GHSP26 present in Gossypium arboreum responds to dehydration. In the present study, an attempt was made to overcome the problem of drought stress in cotton. A cDNA of GHSP26 was isolated from G. arboreum, cloned in plant expression vector, pCAMBIA-1301 driven by the cauliflower mosaic virus 35S promoter and introduced into Gossypium hirsutum. The integration and expression studies of putative transgenic plants were performed through GUS assay; PCR from genomic DNA, and quantitative real-time PCR analysis. Transgenic cotton plants showed an enhanced drought tolerance, suggesting that GHSP26 may play a role in plant responsiveness to drought.

RNAi as a new therapeutic strategy against HCV.

Hepatitis C virus is a major cause of liver associated diseases all over the world. Irrespective of the significant advances in the current therapy, drugs and vaccines are restricted with many factors such as toxicity, complexity, cost and resistance. New technologies particularly RNA interference (RNAi) mediated by small interfering RNA (siRNA) have become more and more interesting and effective therapeutic entities to silence pathogenic gene products associated with disease, including cancer, viral infections and autoimmune disorders. RNAi works at a posttranscriptional level by targeting mRNA as a mean for inhibiting the synthesis of the encoded protein. Several reports have indicated the efficiency and specificity of synthetic and vector based siRNAs inhibiting HCV replication. In the present review, we focused on the recent development in the potential use and issues regarding siRNA as a therapy for HCV.

[Isolation and functional analysis of cotton universal stress protein promoter in response to phytohormones and abiotic stresses].

The 949 bp promoter fragment upstream from the translation initiation site of the GUSP gene encoding a universal stress protein was isolated from the genomic DNA of Gossypium arboream. Some putative cis-acting elements involved in stress responses including E-box, ABRE, DPBF-box, and MYB-core elements were found in the promoter region. In an Agrobacterium-mediated transient expression assay, strong activation of the GUSP full promoter region occurred in tobacco leaves following dehydration, abscisic acid, salt, heavy metal, gibberellic acid and dark treatments. Deletion analysis of the promoter revealed that the dehydration, abscisic acid and salt responses were affected by the deletion between -208 and -949 bp and showed 2-4-fold induction. However, in response to dark, gibberellic acid and heavy metals the induction was only 2-fold. This is an important study as no report of this universal stress protein promoter is available in literature.

Functional analysis of cotton small heat shock protein promoter region in response to abiotic stresses in tobacco using Agrobacterium-mediated transient assay.

The cotton (Gossypium arboreum) stress-related gene GHSP26 responds to dehydration. To elucidate its stress tolerant mechanism at the transcriptional level, we isolated and characterized the promoter region (PGHSP26, -2,831 bp) flanking the 5' GHSP26 coding region from the genomic DNA. A series of PGHSP26 deletion derivatives was created for the identification of the upstream region of the gene required for the promoter activity. Each deletion construct was analyzed by agrobacterium mediated transient transformation in tobacco leaves after treatment with abscissic acid (ABA), heavy metals and dehydration. Promoter fragments of 716 bp or longer showed two-fold or greater induction after each treatment. These findings further our understanding of the regulation of GHSP26 expression and provide a new drought-inducible promoter system in transgenic plants.