Factors affecting protein refolding yields in a fed-batch and batch-refolding system

The refolding of recombinant protein from inclusion bodies expressed in Escherichia coli can present a process bottleneck. Yields at industrially relevant concentrations are restricted by aggregation of protein upon dilution of the denatured form. This article studies the effect of five factors upon the dilution refolding of protein in a twin impeller fed-batch system using refold buffer containing only the oxidized form of the redox reagent. Such a buffer is easier to prepare and more stable than a buffer containing both reduced and oxidized forms. The five factors chosen were: bulk impeller Reynolds number, mini-impeller Reynolds number, injection rate of denatured protein, redox ratio, and guanidine hydrochloride (GdHCl) concentration. A 2(5) factorial experiment was conducted at an industrially relevant protein concentration using lysozyme as the test system. The study identified that in the system used, the guanidine hydrochloride concentration, redox ratio, and injection rate were the most important factors in determining refolding yields. Two interactions were found to be important: redox ratio/guanidine hydrochloride concentration and guanidine hydrochloride concentration/injection rate. Conditions were also found at which high refolding yields could be achieved even with rapid injection and poor mixing efficiency. Therefore, a comparative assessment was carried out with minimal mixing in a simple batch-refolding mode of operation, which revealed different behavior to that of fed-batch. A graphical (windows of operation) analysis of the batch data suggested that optimal yields and productivity are obtained at high guanidine hydrochloride concentrations (1.2 M) and redox ratios of unity or greater.     

The role of phospholipid headgroups in mediating bilayer organization. Perturbations induced by the presence of a tethered chromophore

We report on the dynamics of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (Rhodamine-PE), incorporated into unilamellar vesicles composed of 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC). A key question in the investigation of any bilayer system using tethered fluorescent probes is the role that the chromophore itself plays in determining the organization of phospholipid bilayers. In this work, we investigate the role of headgroup-bound chromophores by measuring the steady state and time-resolved fluorescence response of the tethered rhodamine chromophore as a function of concentration in the bilayer. We find that both the steady state and dynamical properties of the chromophores change with concentration, in a manner consistent with the introduction of disorganization to the bilayers. Steady state fluorescence spectra show a clear perturbation of the rhodamine emission spectrum at a chromophore concentration of 0.25 mol%, which is not seen for lower concentrations, and fluorescence anisotropy data show that both the motional freedom and confining volume experienced by the chromophore increase with concentration. Taken collectively, our data point to the importance of using low concentrations of optical probes in the interrogation of bilayer structures.     

Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-beta. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection.     

Structure of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26 in skimmed milk was found to be composed of d-glucose and d-galactose in a molar ratio of 2:3. Linkage analysis and 1D/2D NMR ((1)H and (13)C) studies performed on the native polysaccharide, and on an oligosaccharide obtained from a partial acid hydrolysate of the native polysaccharide, showed the polysaccharide to consist of branched pentasaccharide repeating units with the following structure. [structure: see text]     

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase. Structural basis for substrate specificity and inactivation by (R)-oxirane-2-carboxylate

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor.     

Chronic hexosamine flux stimulates fatty acid oxidation by activating AMP-activated protein kinase in adipocytes

The hexosamine biosynthesis pathway (HBP) serves as a nutrient sensor and has been implicated in the development of type 2 diabetes. We previously demonstrated that fatty acid oxidation was enhanced in transgenic mouse adipocytes, wherein the rate-limiting enzyme of the HBP, glutamine:fructose-6-phosphate amidotransferase (GFA), was overexpressed. To explore the molecular mechanism of the HBP-induced fatty acid oxidation in adipocytes, we studied AMP-activated protein kinase (AMPK), an energy sensor that stimulates fatty acid oxidation by regulating acetyl-CoA carboxylase (ACC) activity. Phosphorylation and activity of AMPK were increased in transgenic fat pads and in 3T3L1 adipocytes treated with glucosamine to stimulate hexosamine flux. Glucosamine also stimulated phosphorylation of ACC and fatty acid oxidation in 3T3L1 adipocytes, and these stimulatory effects were diminished by adenovirus-mediated expression of a dominant negative AMPK in 3T3L1 adipocytes. Conversely, blocking the HBP with a GFA inhibitor reduced AMPK activity, ACC phosphorylation, and fatty acid oxidation. These changes are not explained by alterations in the cellular AMP/ATP ratio. Further demonstrating that AMPK is regulated by the HBP, we found that AMPK was recognized by succinylated wheat germ agglutinin, which specifically binds O-GlcNAc. The levels of AMPK in succinylated wheat germ agglutinin precipitates correlated with hexosamine flux in mouse fat pads and 3T3L1 adipocytes. Moreover, removal of O-GlcNAc by hexosaminidase reduced AMPK activity. We conclude that chronically high hexosamine flux stimulates fatty acid oxidation by activating AMPK in adipocytes, in part through O-linked glycosylation.     

Phosphorylation of the endogenous thyrotropin-releasing hormone receptor in pituitary GH3 cells and pituitary tissue revealed by phosphosite-specific antibodies

To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr(365) in the receptor tail; distal sites were more phosphorylated in some heterologous models. beta-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nm TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t((1/2)) approximately 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t((1/2)) approximately 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin- and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.