Elucidation of the signalling pathways for enhanced exosome release from Mycobacterium-infected macrophages and subsequent induction of differentiation.

Exosomes are extracellular vesicles released by all cell types; perform several important functions such as cell-to-cell communication, growth, differentiation and so on. Exosomes elicit several signalling mechanisms as they carry information in the form of DNA, RNA or protein docked on them. We show that exosomes released from Mycobacterium tuberculosis (Mtb)-infected macrophages not only induce differentiation of naïve monocytes but also generate functionally active macrophages via MAPK-dependent signalling mechanism through MK-2 and NF-κß activation which is completely different from the differentiation induced by exosomes from uninfected macrophages. Further, we elucidate unequivocally the signalling mechanism behind the enhanced release of exosome generation from infected macrophages driven by AKT phosphorylation involving Rab7a and Rab11a. Genes of both ESCRT-dependent and -independent pathways are found to be involved in enhanced exosomes release and are modulated by AKT. However, interestingly, the genes of the ESCRT-independent pathway are dependent on NF-κß activation while the genes of the dependent pathway are not, suggesting two parallel signalling cascades operating in tandem.

Cloning, soluble expression and purification of high yield recombinant hGMCSF in Escherichia coli.

Expression of human granulocyte macrophage colony stimulating factor (hGMCSF), a cytokine of therapeutic importance, as a thioredoxin (TRX) fusion has been investigated in Escherichia coli BL21 (DE3) codon plus cells. The expression of this protein was low when cloned under the T7 promoter without any fusion tags. High yield of GMCSF was achieved (∼88 mg/L of fermentation broth) in the shake flask when the gene was fused to the E. coli TRX gene. The protein was purified using a single step Ni(2+)-NTA affinity chromatography and the column bound fusion tag was removed by on-column cleavage with enterokinase. The recombinant hGMCSF was expressed as a soluble and biologically active protein in E. coli, and upon purification, the final yield was ∼44 mg/L in shake flask with a specific activity of 2.3 × 10(8) U/mg. The results of Western blot and RP-HPLC analyses, along with biological activity using the TF-1 cell line, established the identity of the purified hGMCSF. In this paper, we report the highest yield of hGMCSF expressed in E. coli. The bioreactor study shows that the yield of hGMCSF could be easily scalable with a yield of ∼400 mg/L, opening up new opportunities for large scale production hGMCSF in E. coli.

A novel prokaryotic vector for identification and selection of recombinants: direct use of the vector for expression studies in E. coli.

BACKGROUND: The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc. RESULTS: We describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the E. coli cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts. CONCLUSIONS: This is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.

Yeast extract mediated autoinduction of lacUV5 promoter: an insight.

We report a simple and cost-effective autoinducible media component responsible for the autoinduction of proteins in Escherichia coli under lacUV5 promoter system. Yeast extract (YE) at high concentration was found to stimulate the expression of T7 RNA polymerase in BL21(DE3) cells while such an effect was not seen in BL21A1 cells. A systematic study on the effect of varying concentrations of YE indicated several folds higher expression of genes viz., human granulocyte colony stimulating factor (rhGCSF), human interferon alpha 2b (rhIFN-alpha2b) and Staphylokinase (rSAK) in BL21(DE3) cells in the absence of any specific inducer like IPTG or additional lactose. Additional investigations on the inducible component of the YE revealed the presence of significant amount of endogenous lactose as the contributory factor for the observed autoinduction phenomenon. This paper highlights the easy scalability of the use of the present media component for large-scale production in biotechnology industry.

Over-expression of proteins using a modified pBAD24 vector in E. coli expression system.

A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine-Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/10(6) c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10(6) c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels.

Leishmania donovani cyclin 1 (LdCyc1) forms a complex with cell cycle kinase subunit CRK3 (LdCRK3) and is possibly involved in S-phase-related activities.

Expression of Leishmania donovani cyclin 1 (LdCyc1) mRNA during the cell cycle of promastigotes is S-phase specific. Here, we show that the LdCyc1 protein is periodically expressed and the activity of its associated kinase varies during the cell cycle in line with its expression pattern. In addition, we have shown that LdCRK3, homologous to CRK3 from L. mexicana, is the cognate Cdk partner of LdCyc1 and that the activity of the complex is inhibited specifically by heat stable factor(s) from the parasite.

Identification, structure, and phylogenetic relationships of a mitogen-activated protein kinase homologue from the parasitic protist Entamoeba histolytica.

A gene encoding mitogen-activated protein kinase (MAPK) from the human enteric parasite, Entamoeba histolytica has been identified. Sequence analyses of the polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) products reveal that the EhMAPK gene is intronless and encodes a protein of 352 amino acids. EhMAPK shows significant homology with other MAPKs and contains the 11 subdomains including the invariant residues characteristic of serine/threonine protein kinases. The MAPK signature residues and motifs are also present in EhMAPK. The atomic model of EhMAPK built with rat ERK2 as template exhibits the conservation of all major secondary structural features. However, a deletion in close proximity to the dual phosphorylation/activation site is of particular interest as it may have functional implications. Phylogenetic analysis indicates that EhMAPK is tightly clustered with Giardia intestinalis ERK2 and Dictyostelium discoideum ERK2. Detailed sequence analysis and phylogenetic study aided us to postulate that EhMAPK belongs to the extracellular signal-regulated kinase (ERK) family. Although EhMAPK bears good homology and phylogenetic closeness with human ERK8 and rat ERK7, sequence analysis indicates that they may be functionally different. The significant differences such as the deletions in the vicinity of the phosphorylation lip, variations in the P+1 specificity pocket, presence of additional acidic amino acids in the common docking domain provide a ground for postulations that activators and substrates for EhMAPK may be to some extent divergent from that of the ERKs of the mammalian host. Although functional characterization of EhMAPK remains to be done, this is the first study of any member of the MAPK signaling system in this organism.

Isolation, characterization and expression of a cyclin from Leishmania donovani.

We have cloned and sequenced a DNA fragment (approximately 1 kb) containing a complete open reading frame from a cDNA library of Leishmania donovani promastigotes. The alignment of the derived polypeptide sequence and the modeling studies revealed that the protein is highly homologous to the mammalian cyclins having conserved cyclin box and substrate-docking motif. Northern blot analysis of the RNA isolated from synchronized L. donovani promastigotes showed periodic expression of the message with maximum abundance at S-phase suggesting its involvement in the events related to the regulation of DNA replication. The results confirm that we have isolated a cyclin molecule from L. donovani (LdCyc1) which may play an important role in the regulation of the parasite cell cycle.