High-Level Expression, Purification and Large-Scale Production of l-Methionine γ-Lyase from Idiomarina as a Novel Anti-Leukemic Drug.

l-Methionine γ-lyase (MGL), a pyridoxal 5'-phosphate-dependent enzyme, possesses anti-tumor activity. However, the low activity of MGL blocks the anti-tumor effect. This study describes an efficient production process for the recombinant MGL (rMGL) from Idiomarina constructed using the overexpression plasmid in Escherichia coli BL21 (DE3), purification, and large-scale production. The enzyme produced by the transformants accounted for 53% of the total proteins and accumulated at 1.95 mg/mL using a 500 L fermentor. The enzyme was purified to approximately 99% purity using a high-pressure mechanical homogenizer and nickel (Ni) Sepharose 6 Fast Flow (FF) chromatography. Then, the enzyme was polished by gel filtration, the endotoxins were removed using diethyl-aminoethanol (DEAE) Sepharose FF, and the final product was lyophilized with a vacuum freeze dryer at -35 °C. The specific activity of rMGL in the lyophilized powder was up to 108 U/mg. Compared to the control, the enzyme significantly inhibited cellular proliferation in a concentration-dependent manner as tested using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and induced cellular apoptosis as analyzed by Annexin V-fluorescein isothiocyanate (FITC) with fluorescence-activated cell sorting (FACS) in leukemia cells. This paper demonstrated the cloning, overexpression, and large-scale production protocols for rMGL, which enabled rMGL to be used as a novel anti-leukemic drug.

[Protective effects of micro-encapsulated Bifidobacteria on gut barrier after hemorrhagic shock and resuscitation: experiment with rats].

OBJECTIVE: To investigate the effects of micro-encapsulated bifidobacteria on gut barrier and bacterial translocation after hemorrhagic shock and resuscitation. METHODS: Sprague-Dawley rats were divided into 6 groups: PBS+sham shock group fed with PBS for 7 days and then undergoing sham shock, bifidobacteria+sham shock group fed with bifidobacteria (10(9) cfu/d) for 7 days and then undergoing sham shock, micro-encapsulated bifidobacteria+sham shock group, fed with micro-encapsulated bifidobacteria (10(9) cfu/d) for 7 days and then undergoing sham shock, PBS+hemorrhagic shock group fed with PBS for 7 days and then undergoing hemorrhagic shock, bifidobacteria+shock group fed with bifidobacteria for 7 days and then undergoing hemorrhagic shock, and micro-encapsulated bifidobacteria+shock group, fed with micro-encapsulated bifidobacteria for 7 days and then undergoing hemorrhagic shock. Three hours after resuscitation laparotomy was performed, distal cecum was resected to undergo bacteriological analysis of the cecal content, mesenteric lymph nodes (MLNs), a liver lobe, and the middle part of spleen were resected to undergo bacterial culture for bacterial translocation, and the terminal ileum was resected to observe the villous damage. RESULTS: There was no significant difference in the amount of blood loss among the 3 hemorrhagic shock groups. The amounts of aerobes in cecum of the bifidobacteria+shock and micro-encapsulated bifidobacteria+shock groups, especially that of the latter group, were significantly lower than that of the PBS+shock group. The amounts of anaerobes and the amounts of bifidobacteria in cecum of the bifidobacteria+shock group and micro-encapsulated bifidobacteria+shock group, especially those of the latter group, were significantly higher than those of the PBS+shock group. No bacterial translocation to liver was observed in all groups. The magnitudes of total aerobes translocation in spleen of the bifidobacteria+shock and encapsulated bifidobacteria+shock groups were significantly lower than that of the PBS+shock group, however, there were not significant differences in the translocation in the MLN of total aerobes ad bifidobacteria among different groups. The percentage of ileal villous damage of the bifidobacteria+shock and encapsulated bifidobacteria+shock groups were significantly lower than that of the PBS+shock group. CONCLUSION: Bifidobacteria effectively protects the gut barrier, reduces bacterial translocation from the gut after hemorrhagic shock and resuscitation. And micro-encapsulated Bifidobacteria can enhance those effects further.