Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

Toxicity Assessment of Cadinene Sesquiterpenes from Eupatorium adenophorum in Mice.

This study evaluated toxic efficacy of Eupatorium adenophorum extracts, against the Kunming mice. In acute study, we firstly tested median lethal dose (LD50) in mice of three cadinene sesquiterpenes 2-deoxo-2-(acetyloxy)-9-oxoageraphorone (DAOA), 9-oxo-agerophorone (OA) and 9-oxo-10,11-dehydro-agerophorone (ODA) from Eupatorium adenophorum (Ea). DAOA (215-4640 mg/kg BW, given orally) showed lowest LD50 at 926 mg/kg BW for male mice in contrast with OA (1470 mg/kg BW) and ODA (1470 mg/kg BW). In sub-acute study, repeated doses (75-300 mg/kg BW, for 7 days) of DAOA/OA increased blood parameters, liver and spleen index in dose dependent relationship, along with decrease in thymus index. The blood biochemical and histopathological examination showed that DAOA/OA dose 300 mg/kg BW significantly causes pathological changes of hepatic lobules and hepatocytes, which are consistent with cholestasis and hepatic injury. 75 mg/kg dose of DAOA/OA was found to be approximately/totally safe over the span of 7 days treatment showing no change in all above described parameters. Cadinene sesquiterpenes guarantee low risk to environment as a type of low toxic botanical components, which may find potential application in biopesticides development field.

Inclusion of quinestrol and 2,6-di-O-methyl-ß-cyclodextrin: Preparation, characterization, and inclusion mode.

An inclusion complex between chemosterilant quinestrol and 2,6-di-O-methyl-ß-cyclodextrin (DM-ß-CD) was prepared using the solution-ultrasonic method. A 1:1 stoichiometry was confirmed by elemental analysis. Analytical techniques such as UV-vis spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, and scanning electron microscopy were used to characterize the complex. Proton NMR and nuclear Overhauser effect spectroscopy results indicate that the hydroxyl end and alkynyl end of quinestrol was included in the DM-ß-CD cavity, which agrees with the most predominant configuration optimized by molecular modeling. The water solubility of quinestrol was significantly increased through complexation with DM-ß-CD. The DM-ß-CD complexes can be used in the design of a novel formulation of quinestrol for rat control products in agriculture.