Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening

In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells coexpressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the reinfection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides

Simultaneous detoxification and preparative separation of chlorogenic acid from Eupatorium adenophorum by combined column chromatography

An efficient method for simultaneous detoxification and preparative separation of chlorogenic acid from Eupatorium adenophorum was developed. The entire procedure consisted of two separation steps. The first step was enrichment of chlorogenic acid from the crude extract by NKA-II macroporous resin. The purity of chlorogenic acid increased from 5.3% to 20.9% with a recovery of 88.7%. Subsequently, polyamide resin was used to achieve a higher purity. After the polyamide resin treatment, the main toxin 9-oxo-10,11-dehydroageraphorone was removed and the purity of chlorogenic acid reached 86.5% with a recovery of 84.8%. It provided a new way for the utilization of E. adenophorum.</p

Ultrasound-assisted aqueous two-phase extraction of phenylethanoid glycosides from Cistanche deserticola Y. C. Ma stems

An efficient ultrasound-assisted aqueous two-phase extraction and enrichment process for phenylethanoid glycosides from Cistanche deserticola Y. C. Ma stems was developed in this work. An ethanol/ammonium sulfate system was chosen for the aqueous two-phase system due to its fine partitioning and recycling behaviors. Single-factorexperiments and response surface methodology were used to optimize the process parameters of the ultrasound-assisted aqueous two-phase extraction. The optimal conditions were as follows: a salt concentration of 23.5%, an ethanol concentration of 20%, an extraction time of 37 min, an extraction temperature of 30 degrees C, a liquid/solid ratio of 30:1 w/w, and an ultrasound power of 300 W. Under the above conditions, the extraction yields of echinacoside and acteoside (the main components of phenylethanoid glycosides) reached 5.35 and 6.22 mg/g dry material weight, respectively. The contents of echinacoside and acteoside in the extracts reached 27.56 and 30.23 mg/g, respectively, which were 2.46- and 2.58-fold higher than the amounts obtained in ultrasound-assisted extraction. In conclusion, ultrasound-assisted aqueous two-phase extraction was an efficient, ecofriendly, and economical method, and it may be a promising technique for extracting and enriching bioactive components from plants

Enrichment and separation of chlorogenic acid from the extract of Eupatorium adenophorum Spreng by macroporous resin

A simple and efficient chromatographic method for separation of chlorogenic acid from Eupatorium adenophorum Spreng extract was developed. The adsorption properties of nine macroporous resins were evaluated. NKA-II resin showed much better adsorption/desorption properties. The adsorption of chlorogenic acid on NKA-II resin at 25 degrees C was well fitted to Langmuir isotherm model and pseudo-second-order kinetic model. The dynamic adsorption and desorption experiments were carried out on columns packed with NKA-II resin to optimize the separation process. The content of chlorogenic acid in the product increased to 22.17%, with a recovery yield of 82.41%. (C) 2015 Elsevier B.V. All rights reserved

Effect of drying methods on physicochemical properties and antioxidant activities of wolfberry (Lycium barbarum) polysaccharide

In this study, an efficient drying process of Lycium barbarum L polysaccharide (LBP) suitable for industrial production was developed and optimized. Three drying methods, including hot air drying (40-80 degrees C), vacuum drying (40-60 degrees C) and spray drying were test and compared. Hot air drying and vacuum drying cost long time and produced a brown product which needs further process due to the agglomeration or alveolation form. The condition of spray drying (without any excipient) was optimized by orthogonal experiment, which gave different optimum conditions based on LBP recovery rate (LBP solution concentration 1.06 g/mL, inlet air temperature 170 degrees C, sample flow rate 15 mL/min and air speed 4.2 m(3)/min) or LBP transparency (LBP solution concentration 1.04 g/mL, inlet air temperature 170 degrees C, sample flow rate 20 mL/min and air speed 2.8 m(3)/min). Pilot scale experiments showed preferable stability of LBP product quality and process parameters. Sample of spray drying (SD) had the highest scavenging free radical effects, the best appearance (LBP transparency), and uniform morphology with hollow sphere which are important properties for the reconstitution of the powder product. Considering the product appearance and product activity, the spray drying was selected to apply in industrial production. (C) 2015 Elsevier Ltd. All rights reserved

Detection of cell populations co-expressing EGFP and DsRed by flow cytometry.

<p>A. Individually infected cells. <i>Sf</i>9 cells individually infected with 1 MOI of vAcBacGFP and vAcBacDsRed were mixed at 48 hpi and used as the reference to gate the two-colored and single-colored cell subsets. B. Cells co-infected with vAcBacGFP and vAcBacDsRed at the MOI of 0.5 each. C. Cells co-infected with 0.8 MOI of vAcBacGFP and 0.2 MOI of vAcBacDsRed. D. Cells co-infected with 0.2 MOI of vAcBacGFP and 0.8 MOI of vAcBacDsRed.</p

Re-infection of <i>Sf</i>9 cells with homologous baculoviruses.

<p><i>Sf</i>9 cells were primarily infected with 1 MOI of vAcBacDsRed, and subsequently infected by 1 MOI of vAcBacGFP at an interval of 1 (top panel), 8 (middle panel) or 16 (bottom panel) hours. Protein expression was monitored 48 hpi of vAcBacDsRed by fluorescent microscopy.</p

Microscopic analysis of <i>Sf</i>9 cells co-infected with vAcBacGFP and vAcBacDsRed.

<p><i>Sf</i>9 cells were simultaneously infected with vAcBacGFP and vAcBacDsRed viruses. Protein expression was observed 48 hpi. Many cells were found co-expressing red and green fluorescent proteins even at low multiplicity. A. Cells were infected with 0.05 MOI of each virus. B. Cells were infected with 0.5 MOI of each virus. C. Cells were infected with vAcBacGFP and vAcBacDsRed at a ratio of 1∶4 respectively. D. Cells were infected with vAcBacGFP and vAcBacDsRed at the ratio of 4∶1 respectively.</p