Efficient production of secretory Streptomyces clavuligerus ß-lactamase inhibitory protein (BLIP) in Pichia pastoris.

ß-Lactamase inhibitory protein (BLIP), a low molecular weight protein from Streptomyces clavuligerus, has a wide range of potential applications in the fields of biotechnology and pharmaceutical industry because of its tight interaction with and potent inhibition on clinically important class A ß-lactamases. To meet the demands for considerable amount of highly pure BLIP, this study aimed at developing an efficient expression system in eukaryotic Pichia pastoris (a methylotrophic yeast) for production of BLIP. With methanol induction, recombinant BLIP was overexpressed in P. pastoris X-33 and secreted into the culture medium. A high yield of ~ 300 mg/L culture secretory BLIP recovered from the culture supernatant without purification was found to be > 90% purity. The recombinant BLIP was fully active and showed an inhibition constant (Ki) for TEM-1 ß-lactamase (0.55 ± 0.07 nM) comparable to that of the native S. clavuligerus-expressed BLIP (0.5 nM). Yeast-produced BLIP in combination with ampicillin effectively inhibited the growth of ß-lactamase-producing Gram-positive Bacillus. Our approach of expressing secretory BLIP in P. pastoris gave 71- to 1200-fold more BLIP with high purity than the other conventional methods, allowing efficient production of large amount of highly pure BLIP, which merits fundamental science studies, drug development and biotechnological applications.

L-lactic acid production by Bacillus subtilis MUR1 in continuous culture.

A novel UV-induced mutant strain of recombinant Bacillus subtilis MUR1 was used for the production of L-LA in continuous cultures with a variety of culture conditions. The maximal productivity of 17.6g/L/h was obtained with a L-LA concentration of 44.1g/L at the dilution rate of 0.4h(-1). The highest concentration of L-LA (77.1g/L) was produced at the dilution rate of 0.05 h(-1). This study showed that the maximum L-LA productivity of B. subtilis MUR1 which can only last for a very short period of time during the exponential phase in fed-batch cultures, can be extended indefinitely at steady state in continuous cultures. L-LA production increased with the increase of yeast extract concentrations in the medium. Moreover, temperature, agitation rate and various glucose concentrations in the feed were compared in continuous cultures. Different nitrogen sources (lysine, glutamine, ammonium sulphate and corn steep liquor) were studied to partly or completely replace yeast extract in the medium, most of them showed positive effects on L-LA production and cell growth. The L-LA productivities from continuous cultures in this study are higher than the productivity of current microbial industrial processes which use Lactobacillus to produce L-LA.

L-lactic acid production by Bacillus subtilis MUR1.

Bacillus subtilis MUR1 is a novel lactic acid (LA) producing strain that has the potential for industrial production of LA due to its high productivity of LA, high yield of substrate conversion, and high final concentration of LA produced. B. subtilis MUR1 can produce 99.3 and 183.2g/l of L-LA in 12 and 52 h respectively with a 98.5% substrate conversion yield and a maximum L-LA production rate of 16.1g/l/h. Compared with batch culture, and several fed-batch cultures with different initial glucose concentrations, the fed-batch culture with initial 30 g/l glucose produced the highest final concentration and productivity of L-LA. Corn steep liquor can be used to partly replace yeast extract in the production medium for the production of L-LA by B. subtilis MUR1.

Development and iron-dependent expression of hephaestin in different brain regions of rats.

It has been suggested that Hephaestin (Heph), a newly discovered ceruloplasmin homologue, is necessary for iron egress from the enterocytes into circulation via interacting with ferroportin1 (FP1). Based on the putative function of Heph, and the similarity between the process of iron transport in the enterocytes and that in the blood-brain barrier (BBB) cells, it has also been proposed that Heph plays a similar role in exporting iron from the BBB cells and other brain cells as it works in the enterocytes via interacting with FP1. The existence of FP1 in the brain has been demonstrated. In this study, we investigated Heph expression and effects of development and iron in the cortex, hippocampus, striatum, and substantia nigra. The data demonstrated that all the four regions we examined have the ability to express Heph mRNA and protein. The findings also showed that both the development and iron status have a significant effect on Heph expression and the effects of iron status are regionally specific. It was also suggested that Heph expression is probably regulated at the transcriptional level by the development and iron in these brain regions. These findings, together with other published data, support a putative role of Heph in the iron metabolism in the brain.

Role of soluble ceruloplasmin in iron uptake by midbrain and hippocampus neurons.

Ceruloplasmin (CP) is essential for brain iron homeostasis. However, its precise function in brain iron transport has not been definitely determined. In this study, we investigated the effects of soluble CP on iron influx and efflux in primary neuronal culture from the midbrain (the substantia nigra and striatum) and the hippocampus. Our data showed that low concentrations of CP (2, 4, 8 microg/ml) can promote iron influx into iron-deficient neurons, but not the neurons with normal iron status. The same concentrations of CP had no effect on iron efflux from iron-sufficient and normal-iron neurons. Contrary to our expectation, we did not find any regional difference in the effects of CP on iron influx as well as efflux in neurons. The changes in quenching (iron influx) and also dequenching (iron efflux) of intracellular fluorescence, induced by the addition of CP with iron, in the midbrain neurons were no different from those in the hippocampus neurons. The data showed that soluble CP has a role in iron uptake by iron-deficient brain neurons under our experimental conditions. The physiological significance of the results forms the focus for future work.

Increased divalent metal transporter 1 expression might be associated with the neurotoxicity of L-DOPA.

Based on the available data, we speculated that changes in brain iron metabolism induced by L-DOPA might be associated with the neurotoxicity of L-DOPA. To investigate this possibility, the effects of L-DOPA on the expression of iron influx proteins [transferrin receptor (TfR) and divalent metal transporter 1 (DMT1)], iron efflux protein (ferroportin 1), and iron uptake in C6 glioma cells were determined in this study using Northern blot and Western blot analysis and the calcein method. The findings showed that treatment of C6 cells with different concentrations of L-DOPA (0-100 microM) did not affect the expression of mRNA and protein of TfR and DMT1 with iron-responsive element (+IRE) and protein of ferroportin 1. However, a significant increase in the expression of DMT1(-IRE) mRNA and protein was found in cells treated, respectively, with 10 and 30 microM L-DOPA (mRNA) and 1, 5, 10 and 30 microM L-DOPA (protein). The increase in DMT(-IRE) protein induced by L-DOPA treatment was in parallel with the increase in DMT(-IRE) mRNA. The levels of DMT1(-IRE) mRNA and protein peaked in the cells treated with 10 microM L-DOPA and then decreased progressively with increasing concentrations of L-DOPA. Further study demonstrated that treatment of the cells with 10 microM L-DOPA induced a significant increase in ferrous uptake by C6 glioma cells. The findings suggested that the increased DMT1(-IRE) expression might be partly associated with the neurotoxicity of L-DOPA. Clinical relevance of the findings needs to be investigated further.

[Optimization of the expression of human DNA topoisomerase I in Pichia pastoris].

Human DNA Topoisomerase I (hTopo I) has been identified to be an efficient target of many effective antitumor drugs. Natural hTopo I is not convenient to be used in screening because of its low concentration in cells. In order to fast screen new anticancer drugs targeting at hTopo I from natural compounds in vitro, hTopo I gene open reading frame (ORF) has been successfully cloned and overexpressed in Pichia pastoris. Total RNA extracted from Hela cells was reversely transcripted to synthesize cDNA with the hTopo I specific antisense primer and the hTopo I ORF was synthesized by PCR. After digestion with EcoR I and Kpn I, the synthesized fragment was inserted into pPICZaA, gave rise to pPICZalpha-hTopoI. After digestion with Sac I, the lined pPICZalpha-hTopoI was transformed into Pichia pastoris strains (KM71, X33 and SMD1168) by electroporation and integrated into their genome. After screened on YPDS plates (containing 1000 ug/mL zeocin), the high-copy recombinant strains (KM-hTopoI, X33-hTopoI and SMD-hTopol) could overexpress recombinant hTopo I, which was fused to the alpha-factor secretion signal and could be secreted into the supernatant in the culture. alpha-factor could be cleaved from the expressed protein during secretion. A higher activity amount of the enzyme was secreted by the particular strain SMD-hTopoI because of its absence of proteimase A than by other strains which possess proteinase A activity. After optimizing the fermentation conditions, a relatively higher enzyme activity in the culture supernatant could be obtained when SMD-hTopoI was induced in BMMY (pH7.25) at 20 degrees C , with addition of 0.5% (V/V) methanol and 3% (V/V) nutrient liquid every 24h. The enzyme activity reached 43 000 u/mL, the yield reached 11 mg/L, achieving approximate 10% of total protein in the culture supernatant. SDS-PAGE and Western blot analyses showed that the mass of the recombinant hTopo I was 91 kD with no glycosylation.

Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway.

The membrane transferrin receptor-mediated endocytosis or internalization of the complex of transferrin bound iron and the transferrin receptor is the major route of cellular iron uptake. This efficient cellular uptake pathway has been exploited for the site-specific delivery not only of anticancer drugs and proteins, but also of therapeutic genes into proliferating malignant cells that overexpress the transferrin receptors. This is achieved either chemically by conjugation of transferrin with therapeutic drugs, proteins, or genetically by infusion of therapeutic peptides or proteins into the structure of transferrin. The resulting conjugates significantly improve the cytotoxicity and selectivity of the drugs. The coupling of DNA to transferrin via a polycation or liposome serves as a potential alternative to viral vector for gene therapy. Moreover, the OX26 monoclonal antibody against the rat transferrin receptor offers great promise in the delivery of therapeutic agents across the blood-brain barrier to the brain.