Soluble expression of recombinant nerve growth factor in cytoplasm of escherichia coli

Background: Pivotal roles of Nerve growth factor [NGF] in the development and survival of both neuronal and non-neuronal cells indicate its potential for the treatment of neurodegenerative diseases. However, investigation of NGF deficits in different diseases requires the availability of properly folded human beta-NGF. In previous studies bacterial expression of hNGF demonstrated the feasibility of its overproduction. However, known limitations in the use of E. coli as an expression host for a protein with three intra-chain disulfide bonds were evident

Objectives: Here an optimized system was developed to overexpress the soluble NGF in E. coli

Materials and Methods: The gene encoding the beta subunit of mature hNGF was optimized based on E. coli codon preference and cloned into pET-32a expression vector providing His- and Trx- tags for detection and increasing the solubility of recombinant protein, respectively. The recombinant DNA was expressed in E. coli Origami [DE3], which enhances the correct formation of disulfide bonds in the cytoplasm of E. coli. Different culture conditions were evaluated to increase soluble expression of the target protein

Results: The highest soluble expression level was achieved when E. coli Origami [DE3] cells expressing NGF were grown at 30[degree]C in TB medium with 0.2 mM IPTG induction at OD[600nm] = 1 for 4 h

Conclusions: Our results indicated that the recombinant NGF was successfully expressed as a soluble form

Tissue specific expression of human calcitonin gene in potato tubers by an organ specific promoter

To increase the production level of heterologous proteins in plants, strategies such as choice of stronger promoters, optimization of codon usage and specific localization of foreign proteins are of major concern. Calcitonin [CT], a 32 amino acid polypeptide is a powerful and specific inhibitor of bone resorption and is used to treat several human diseases. Calcitonin activity is not species-specific which make it possible to produce in various animal sources, however, antibody formation in the prolonged application of animal CT leads to gradual decrease or loss of activity. That is why the long term treatment of human patients with CT requires homologous calcitonin. In this study, a human calcitonin [hCT] gene, driven by two different promoters [granule bound starch synthase I and Cauliflower mosaic virus 35S] was expressed in potato plants, using Agrobacterium-mediated transformation. Molecular analysis, including PCR, RT-PCR, Northern dot blot hybridizations showed that hCT could be successfully transcribed in transgenic potato plants. The immunoassay results showed that tissue specific expression in potato, led to almost five-fold more hCT accumulation than constitutively expression in all plant tissues

In silico genome-wide screening for TnrA-regulated genes of Bacillus clausii

Bacillus clausii TnrA transcription factor is required for global nitrogen regulation. In order to obtain an overview of gene regulation by TnrA in B. clausii KSM-K16, the entire genome of B. clausii was screened for the consensus sequence, 5'-TGTNAN7TNACA-3' known as the TnrA box, and 13 transcription units were found containing a putative TnrA box. The TnrA targets identified in this study were tnrA, glnA, nrgA, nasFDEB, puc genes, licT, the two operons of the oligopeptide ABC transporter, lytR, transcriptional regulator of the Lrp/AsnC family, sodium-dependent transporter of SNF family, hyu genes and a biochemically uncharacterized protein