Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in <it>E. coli </it>frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant <it>E. coli</it>. Here, we describe the successful use of the immobilized folding machineries for <it>in vitro </it>refolding with the examples of high yield refolding of a ribonuclease A (RNase A) and cyclohexanone monooxygenase (CHMO).</p> <p>Results</p> <p>We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191–345 of GroEL) and two foldases (DsbA and human peptidyl-prolyl <it>cis-trans </it>isomerase) by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively.</p> <p>Conclusion</p> <p>The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of <it>E. coli </it>inclusion bodies in high yield with biological function.</p

pH-responsive high-density lipoprotein-like nanoparticles to release paclitaxel at acidic pH in cancer chemotherapy

Jae-Yoon Shin,1,* Yoosoo Yang,1,* Paul Heo,1 Ji-Chun Lee,1 ByoungJae Kong,1 Jae Youl Cho,1 Keejung Yoon,1 Cheol-Su Shin,2 Jin-Ho Seo,3 Sung-Gun Kim,4 Dae-Hyuk Kweon11Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University, 2APTech Research Center, Suwon, 3Department of Agricultural Biotechnology, Seoul National University, Seoul, 4Department of Biomedical Science, Youngdong University, Chungbuk, South Korea*These authors contributed equally to this workBackground: Nanoparticles undergoing physicochemical changes to release enclosed drugs at acidic pH conditions are promising vehicles for antitumor drug delivery. Among the various drug carriers, high-density lipoprotein (HDL)-like nanoparticles have been shown to be beneficial for cancer chemotherapy, but have not yet been designed to be pH-responsive.Methods and results: In this study, we developed a pH-responsive HDL-like nanoparticle that selectively releases paclitaxel, a model antitumor drug, at acidic pH. While the well known HDL-like nanoparticle containing phospholipids, phosphatidylcholine, and apolipoprotein A-I, as well as paclitaxel (PTX-PL-NP) was structurally robust at a wide range of pH values (3.8&amp;ndash;10.0), the paclitaxel nanoparticle that only contained paclitaxel and apoA-I selectively released paclitaxel into the medium at low pH. The paclitaxel nanoparticle was stable at physiological and basic pH values, and over a wide range of temperatures, which is a required feature for efficient cancer chemotherapy. The homogeneous assembly enabled high paclitaxel loading per nanoparticle, which was 62.2% (w/w). The molar ratio of apolipoprotein A-I and paclitaxel was 1:55, suggesting that a single nanoparticle contained approximately 110 paclitaxel particles in a spherical structure with a 9.2 nm diameter. Among the several reconstitution methods applied, simple dilution following sonication enhanced the reconstitution yield of soluble paclitaxel nanoparticles, which was 0.66. As a result of the pH responsiveness, the anticancer effect of paclitaxel nanoparticles was much more potent than free paclitaxel or PTX-PL-NP.Conclusion: The anticancer efficacy of both paclitaxel nanoparticles and PTX-PL-NP was dependent on the expression of scavenger receptor class B type I, while the killing efficacy of free paclitaxel was independent of this receptor. We speculate that the pH responsiveness of paclitaxel nanoparticles enabled efficient endosomal escape of paclitaxel before lysosomal break down. This is the first report on pH-responsive nanoparticles that do not contain any synthetic polymer.Keywords: pH responsiveness, nanoparticle, apolipoprotein A-I, paclitaxe

Effect of mucilage from yam on activation of lymphocytic immune cells

The immunostimulating activities of mucilage fraction from yam were investigated. The proliferation of BSA-primed lymph node cells was enhanced between 4.1- to 10.9-fold compare to control, when cultured with 1 to 25 µg/mL of yam-mucilage fraction. It showed strong immunopotentiating activity than ginseng extract and as remarkable as Bifidobacterium adolescentis M101-4 known as a positive immunostimulator. Mitogenicity to lymph node cells was fully induced by concanavalin A and lipopolysaccharide. The proliferation of splenocytes and Peyer's patch cells was enhanced between 5.0- to 14.1-fold and 2.4- to 6.4-fold, respectively, when cultured with 1 to 25 µg/mL of yam-mucilage fraction. It enhanced the production of cytokines such as tumor necrosis factor-α and IL-6 in the culture of RAW 264.7 macrophage cells. In the culture of lipopolysaccharide-stimulated RAW 264.7 cells, production of cytokines was as similar as compared to controls. In unstimulated RAW 264.7 cells, both tumor necrosis factor-α and IL-6 production were enhanced between 15.6- to 60.1-fold and 2.3- to 9.1-fold, respectively. Mucilage fraction from yam is expected to be a safe immunopotentiator to maintain the host immunity and develop a physiologically functional food

Genetic analysis of ORF5 of recent Korean porcine reproductive and respiratory syndrome viruses (PRRSVs) in viremic sera collected from MLV-vaccinating or non-vaccinating farms

The 23 open reading frame (ORF) 5 sequences of Korean type II porcine reproductive and respiratory syndrome virus (PRRSV) were collected from viremic sera from the (modified live vaccine) MLV-vaccinating and non-vaccinating farms from 2007 to 2008. The samples were phylogenetically analyzed with previous ORF5 sequences, including type I Korean PRRSV, and previously reported or collected sequences from 1997 to 2008. A MN184-like subgroup of type II Korean PRRSV was newly identified in the viremic sera collected from 2007 to 2008. And of the type I PRRSVs, one subgroup had 87.2~88.9% similarity with the Lelystad virus, showing a close relationship with the 27~2003 strain of Spain. The maximum parsimony tree of type II PRRSV from 1997 to 2008 showed that they had evolved to four lineages, subgroups 1, 2, 3 and 4. Most of the recently collected type II PRRSVs belonged to subgroup 4 (48%). The region of three B-cell epitopes and two T-cell epitopes of ORF5 amino acids sequences was considerably different from the MLV in subgroups 3 and 4. In conclusion, the existence of type I PRRSV, which was genetically different from Lelystad virus (Prototype of type I PRRSV), and heterologous type II PRRSVs of viremic pigs detected even in the MLV-vaccinating farms indicated the need for new vaccine approaches for the control of PRRSV in Korea

Chlorogenic Acid Isomers Isolated from <i>Artemisia lavandulaefolia</i> Exhibit Anti-Rosacea Effects In Vitro

Rosacea is a chronic inflammatory disease affecting facial skin. It is associated with immune and vascular dysfunction mediated via increased expression and activity of cathelicidin and kallikrein 5 (KLK5), a serine protease of stratum corneum. Therefore, KLK5 inhibitors are considered as therapeutic agents for improving the underlying pathophysiology and clinical manifestation of rosacea. Here, we isolated the active constituents of Artemisia lavandulaefolia (A. lavandulaefolia) and investigated their inhibitory effect on KLK5 protease activity. Using bioassay-guided isolation, two bioactive compounds including chlorogenic acid isomers, 3,5-dicaffeoylquinic acid (isochlorogenic acid A) (1), and 4,5-dicaffeoylquinic acid (isochlorogenic acid C) (2) were isolated from A. lavandulaefolia. In this study, we evaluated the effects of isochlorogenic acids A and C on dysregulation of vascular and immune responses to rosacea, and elucidated their molecular mechanisms of action. The two chlorogenic acid isomers inhibit KLK5 protease activity, leading to reduced conversion of inactive cathelicidin into active LL-37. This inhibition of LL-37 production by isochlorogenic acids A and C reveals the efficacy of suppressing the expression of inflammatory mediators induced by LL-37 in immune cells such as macrophages and mast cells. In addition, both isomers of chlorogenic acid directly inhibited the proliferation and migration of vascular endothelial cells induced by LL-37