Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g⁻¹, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l⁻¹, resulted in a dramatic increase in volumetric yield (85,600 U l⁻¹) and global volumetric productivity (1083 U l⁻¹ h⁻¹).     

Virtual screening and biophysical studies lead to HSP90 inhibitors

Heat shock protein 90 (HSP90) is a molecular chaperone that plays important functional roles in cells. The chaperone activity of HSP90 is regulated by the hydrolysis of ATP at the protein’s N-terminal domain. HSP90, in particular the N-terminal domain, is a current inhibition target for therapeutic treatments of cancers. This paper describes an application of virtual screening, thermal shift assaying and protein NMR spectroscopy leading to the discovery of HSP90 inhibitors that contain the resorcinol structure. The resorcinol scaffold can be found in a class of HSP90 inhibitors that are currently undergoing clinical trials. The proved success of the resorcinol moiety in HSP90 inhibitors validates this combined virtual screen and biophysical technique approach, which may be applied for future inhibitor discovery work for HSP90 as well as other targets.     

Biotechnological significance of toxic marine dinoflagellates

Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review.     

IDENTIFICATION OF CULTURED PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) USING SPECIES-SPECIFIC LSU rRNA-TARGETED FLUORESCENT PROBES1

Some, but not all, marine pennate diatoms of the genus Pseudo-nitzschia H. Peragallo are associated with the production of domoic acid, a naturally occurring amino acid responsible for amnesic shellfish poisoning. Distinguishing between potentially toxic and nontoxic representatives of this genus is time-consuming and difficult because it demands scanning electron microscopy of cleaned frustules. The objective of this work is to speed and ease identification of these organisms by using whole-cell (in situ) hybridization and species-specific large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotide probes. Toward that end, cultures of P. australis Frenguelli, P. pungens (Grunow) Hasle, P. multiseries (Hasle) Hasle, P. fraudulenta (P. T. Cleve) Heiden, P. heimii Manguin, P. delicatissima (P. T. Cleve) Heiden, P. pseudo-delicatissima (Hasle) Hasle, and P. americana (Hasle) Fryxell were screened with a suite of 15 putative species-specific probes. Of those, a subset of eight probes was found that distinguished each species tested. In addition, Pseudo-nitzschia chloroplasts were labeled with a probe directed against a eubacterial-conserved sequence. Identification of new cultures based on their reactivity toward a set of probes agreed with species designations as defined by morphological criteria. Whole-cell hybridization is a rapid, simple, and cost-effective technique for discriminating among cultured Pseudo-nitzschia species.     

Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase

A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.     

PSEUDO-NITZSCHIA SPECIES (BACILLARIOPHYCEAE) IN LOUISIANA COASTAL WATERS: MOLECULAR PROBE FIELD TRIALS, GENETIC VARIABILITY, AND DOMOIC ACID ANALYSES

An 18-month field survey of the Pseudo-nitzschia population present in Louisiana coastal waters was conducted comparing species abundance estimates by novel fluorescent molecular probes (16S large subunit rDNA oligonucleotide sequences) with traditional electron and differential-interference light microscopy. While the probe and microscopic analyses agreed on the presence or absence of four common Pseudo-nitzschia species ( P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, P. delicatissima (P.T. Cleve) Heiden, and P. pungens (Grunow) Hasle in 66% of the samples analyzed, the probes gave conflicting results with the microscopic methods in the remaining 34% of the samples. The majority of the discrepancies appear to be because of genetic variation within the Pseudo-nitzschia population, especially in P. pseudodelicatissima, indicating that the Monterey Bay Pseudo-nitzschia spp. may not be appropriate reference strains for distinguishing Louisiana Pseudo-nitzschia spp. Additionally, P. pseudodelicatissima has been associated with domoic acid (DA) activity in three field samples, at levels up to 22 times higher than the highest value given inother published reports of DA production by this species. The contemporaneous existence of multiple strains of P. pseudodelicatissima (toxic and non-toxic) presents new challenges to the study of the ecophysiology and population dynamics of this bloom-forming species.     

Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability

We engineered nucleosome core particles (NCPs) with two site-specific cysteine crosslinks that increase the stability of the particle. The first disulfide was introduced between the two copies of H2A via an H2A-N38C point mutation, effectively crosslinking the two H2A/H2B heterodimers together to stabilize the histone octamer against H2A/H2B dimer dissociation. The second crosslink was engineered between an R40C point mutation on the N-terminal tail of H3 and the NCP DNA ends by the introduction of a convertible nucleotide. This crosslink maintains the nucleosome DNA in a fixed translational setting relative to the histone octamer and prevents dilution-driven dissociation. The X-ray crystal structures of NCPs containing the disulfides in isolation and in combination were determined. Both disulfides stabilize the structure of the NCP without disturbing the overall structure. Nucleosomes containing these modifications will be advantageous for biochemical and structural studies as a consequence of their greater resistance to dissociation during high dilution in purification, elevated salt for crystallization and vitrification for cryogenic electron microscopy.     

Analysis and identification of essential genes in humans using topological properties and biological information

Genes that are indispensable for survival are termed essential genes. The analysis and identification of essential genes are very important for understanding the minimal requirements of cellular survival and for practical purposes. Proteins do not exert their function in isolation of one another but rather interact together in PPI networks. A global analysis of protein interaction networks provides an effective way to elucidate the relationships between proteins. With the recent large-scale identifications of essential genes and the production of large amounts of PPIs in humans, we are able to investigate the topological properties and biological properties of essential genes. However, until recently, no one has ever investigated human essential genes using topological and biological properties. In this study, for the first time, 28 topological properties and 22 biological properties were used to investigate the characteristics of essential and non-essential genes in humans. Most of the properties were statistically discriminative between essential and non-essential genes. The F-score was used to estimate the essentiality of each property. The GO-enrichment analysis was performed to investigate the functions of the essential and non-essential genes. Finally, based on the topological features and the biological characteristics, a machine-learning classifier was constructed to predict the essential genes. The results of the jackknife test and 10-fold cross validation test are encouraging, indicating that our classifier is an effective human essential gene discovery method.