Cloning,Expression, and Characterization of Rice α-Galactosidase

We have successfully cloned an α-galactosidase gene from a rice cDNA library and transformed it into Escherichia coli BL21. It was subsequently cloned to the pPIC9K vector and expressed in Pichia pastoris. A selected clone was found to result in high production yield of the galactosidase enzyme. The secreted enzyme was purified, and it revealed as a major protein band on an SDS-PAGE gel. The optimal pH value, enzyme stabilities, and substrate specificity were studied. The enzyme specificity toward the terminal α1→6, 1→4, and 1→3 linked galactosyl residue from various substrates was investigated. By determining the Michelis constant (Km) of the enzyme for melibiose, raffinose, and stachyose, our results showed that melibiose was hydrolyzed faster than raffinose, whereas the published data reported a reversed sequence, raffinose > melibiose. The enzyme also showed the ability of converting B red blood cells into O red cells. The objective of this work is to develop the Pichia system to produce a large quantity of enzyme for blood cell conversion for transfusion.     

High-Yield Expression, Purification, and Characterization of Active, SolubleBacteroides fragilisMetallo-β-Lactamase, CcrA

The gene fromBacteroides fragilisencoding a metallo-β-lactamase,ccrA,was expressed inEscherichia coliBL21(DE3) containing the wild-type disulfide bond-catalyzing systemdsbas an active, soluble enzyme in quantities exceeding 100 mg/liter using both rich and minimal media. Both the nonfusion and a glutathioneS-transferase fusion enzyme lacking the periplasmic signal sequence were purified to homogeneity. Characteristics of the purified nonfusion enzyme are shown to be similar to those of the renatured enzyme previously reported. Thermal denaturation studies using circular dichroism and fluorescence spectroscopy show that CcrA undergoes a transition at ∼50°C which corresponds to the transition temperature of catalytic activity. The secondary structure of the protein and the catalytic apparatus are thus intimately linked.     

Calcium Channel α2δ Subunits: Differential Expression, Function, and Drug Binding

Voltage-activated calcium channels are transmembrane proteins that act as transducers of electrical signals into numerous intracellular activities. On the basis of their electrophysiological properties they are classified as high- and low-voltage-activated calcium channels. High-voltage-activated calcium channels are heterooligomeric proteins consisting of a pore-forming alpha1 subunit and auxiliary alpha2delta, beta, and--in some tissues--gamma subunits. Auxiliary subunits support the membrane trafficking of the alpha1 subunit and modulate the kinetic properties of the channel. In particular, the alpha2delta subunit has been shown to modify the biophysical and pharmacological properties of the alpha1 subunit. The alpha2delta subunit is posttranslationally cleaved to form disulfide-linked alpha2 and, delta proteins, both of which are heavily glycosylated. Recently it was shown that at least four genes encode for alpha2delta subunits which are expressed in a tissue-specific manner. Their biophysical properties were characterized in coexpression studies with high- and low-voltage-activated calcium channels. Mutations in the gene encoding alpha2delta-2 have been found to underlie the ducky phenotype. This mouse mutant is a model for absence epilepsy and is characterized by spike wave seizures and cerebellar ataxia. Alpha2delta subunits can also support pharmacological interactions with drugs that are used for the treatment of epilepsy and neuropathic pain.     

Cloning,Expression, and Characterization of a Peculiar Choline-Binding β-Galactosidase from Streptococcus mitis

A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric β-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40°C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In S. mitis, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first β-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.Streptococcus mitis belongs to the viridans group of streptococci and is a relevant microorganism because it is both an opportunistic pathogen and phylogenetically close to Streptococcus pneumoniae, a major respiratory human pathogen. Although S. mitis isolates usually produce only mild infections, some S. mitis strains have acquired increased virulence and are one of the main causes of infectious endocarditis (15, 36). Remarkably, S. mitis, like only a few other streptococci, displays phosphorylcholine residues in its cellular envelope (3). This aminoalcohol is used for the anchorage of proteins belonging to the so-called “choline-binding proteins” (CBPs), which fulfill important physiological functions in these bacteria. CBPs bind to phosphorylcholine residues present in the teichoic and lipoteichoic acids located at the surface of S. pneumoniae and some streptococci of the mitis group. CBPs share a modular organization consisting of a biologically active domain and a conserved choline-binding domain (CBD), which contains 6 to 18 imperfect 20-amino-acid tandem repeats each located either at the carboxy- or amino-terminal ends of the proteins (26). This CBD is able to specifically bind to choline or its structural analogues like DEAE (diethylaminoethanol), which permits purification by affinity chromatography in a single step using DEAE-cellulose supports (38). Crystallographic studies of CBPs have shown that a typical CBD consists of several β-hairpins organized as a left-handed superhelix and that the linkage of CBPs to the choline-containing cell wall substrate is carried out through the binding of choline residues to the interface of two consecutive choline-binding repeats, named choline-binding sites (9, 13, 14).β-d-Galactosidases (β-d-galactoside galactohydrolase; EC 3.2.1.23) constitute a large family of proteins that cleave the glycosidic bond between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety, e.g., lactose and related chromogens, like o-nitrophenyl-β-d-galactopyranoside (ONPG), p-nitrophenyl-β-d-galactopyranoside (PNPG), or 6-bromo-2-naphthyl-galactopyranoside. β-d-galactosidases belong to the glycosyl hydrolase (GH) superfamily, which contains 114 families (see http://www.CAZY.org) classified on the basis of amino acid sequence similarity (12). The enzymes exhibiting β-galactosidase activity are currently classified within four different families: GH-1, GH-2, GH-35, and GH-42. β-Galactosidases are widely distributed in nature and are present in numerous microorganisms (yeasts, fungi, bacteria, and archaea), plants, and animals (34, 44). These enzymes are of great interest for several industrial or biotechnological processes; the hydrolytic activity has been applied in the food industry for decades to reduce the lactose content of milk products in order to circumvent lactose intolerance, which is prevalent in more than half of the world''s population (27). More recently, interest in β-galactosidases has increased due to their ability to synthesize β-galactosyl derivatives that have received a great deal of attention owing to their important roles in many biological processes (27).In this study, we report the purification and biochemical characterization of a peculiar β-galactosidase encoded by the SMT1224 gene of S. mitis that represents a new type of β-galactosidase within this paradigmatic enzyme family.     

Sex Expression in Flowering

Sex expression constitutes a specific aspectof the physiology of flowering and provides agood subject for the study of differentiation.Although most plants are hermaphroditic, quitea number of them are monoecious or dioecious,in which one is faced with the specific problemof sex expression beyond the general problem offlowering. Our earlier work on this subject can be tra-ced back to 1957 (Tsao et al. 1957).Earlier work on potted plants In pot experiments, we have studied the     

Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase

Biosynthesis of repeat-unit polysaccharides and N-linked glycans proceeds by sequential transfer of sugars from the appropriate sugar donor to an activated lipid carrier. The transfer of each sugar is catalysed by a specific glycosyltransferase. The molecular basis of the specificity of sugar addition is not yet well understood, mainly because of the difficulty of isolating these proteins. In this study, the aceA gene product expressed by Acetobacter xylinum, which is involved in the biosynthesis of the exopolysaccharide acetan, was overproduced in Escherichia coli and its function was characterised. The aceA ORF was subcloned into the expression vector pET29 in frame with the S·tag epitope. The recombinant protein was identified, and culture conditions were optimised for production of the soluble protein. The results of test reactions showed that AceA is able to transfer one α-mannose residue from GDP-mannose to cellobiose-P-P-lipid to produce α-mannose-cellobiose-P-P-lipid. AceA was not able to use free cellobiose as a substrate, indicating that the pyrophosphate-lipid moiety is needed for enzymatic activity. Received: 11 February 1999 / Accepted: 6 April 1999     

Expression,Purification, and Characterization of Recombinant Human α<Subscript>1</Subscript>-Antitrypsin Produced Using Silkworm–Baculovirus Expression System

Human α₁-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α₁-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~?15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.     

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α

POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.     

Design, Recombinant Expression, and Antibacterial Activity of the Cecropins–Melittin Hybrid Antimicrobial Peptides

In order to evaluate their antibacterial activities and toxicities, the cecropins–melittin hybrid antimicrobial peptide, CA(1-7)-M(4-11) (CAM) and CB(1-7)-M(4-11) (CBM), were designed by APD2 database. The recombinant hybrid antimicrobial peptides were successfully expressed and purified in Pichia pastoris. Antimicrobial activity assay showed that both of the two hybrid antimicrobial peptides had strong antibacterial abilities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Bacillus thuringiensis, and Salmonella derby. The potency of CAM and CBM to E. coli 25922 were 0.862 and 0.849, respectively, slightly lower than Amp’s 0.957. The hemolytic assays indicated CAM and CBM had no hemolytic in vivo and in vitro, and so they had a good application prospect.     

Expression,Purification, and Evaluation of Anti-IL-2Rα Antibody Secreted by Leishmania tarentolae

International Journal of Peptide Research and Therapeutics - Producing a functional anti-IL-2Rα antibody in Leishmania tarentolae, a trypanosomatid protozoan non-pathogenic to human, is a...     

mTORC1 Hyperactivity Inhibits Serum Deprivation-Induced Apoptosis via Increased Hexokinase II and GLUT1 Expression,Sustained Mcl-1 Expression,and Glycogen Synthase Kinase 3β Inhibition

The current concept is that Tsc-deficient cells are sensitized to apoptosis due to the inhibition of Akt activity by the negative feedback mechanism induced by the hyperactive mTORC1. Unexpectedly, however, we found that Tsc1/2-deficient cells exhibit increased resistance to serum deprivation-induced apoptosis. mTORC1 hyperactivity contributes to the apoptotic resistance of serum-deprived Tsc1/2-deficient cells in part by increasing the growth factor-independent expression of hexokinase II (HKII) and GLUT1. mTORC1-mediated increase in hypoxia-inducible factor 1α (HIF1α) abundance, which occurs in the absence of serum in normoxic Tsc2-deficient cells, contributes to these changes. Increased HIF1α abundance in these cells is attributed to both an increased level and the sustained translation of HIF1α mRNA. Sustained glycogen synthase kinase 3β inhibition and Mcl-1 expression also contribute to the apoptotic resistance of Tsc2-deficient cells to serum deprivation. The inhibition of mTORC1 activity by either rapamycin or Raptor knockdown cannot resensitize these cells to serum deprivation-induced apoptosis because of elevated Akt activity that is an indirect consequence of mTORC1 inhibition. However, the increased HIF1α abundance and the maintenance of Mcl-1 protein expression in serum-deprived Tsc2^−/− cells are dependent largely on the hyperactive eIF4E in these cells. Consistently, the reduction of eIF4E levels abrogates the resistance of Tsc2^−/− cells to serum deprivation-induced apoptosis.Growth factors are obligatory for the survival of mammalian cells. The evolutionarily conserved kinase Akt has emerged as the predominant and indispensable mediator of the ability of growth factors to promote cell survival in mammalian cells (reviewed in reference 9). Akt promotes cell survival by multiple mechanisms, including key roles in regulating cellular energy metabolism. Akt maintains mitochondrial integrity and inhibits apoptosis at least in part through effects on mitochondrial hexokinases and their functionally coupled facilitated glucose transporters (reviewed in reference 18). One of the most crucial functions of Akt involves the activation of the mammalian target of rapamycin complex 1 (mTORC1), which integrates growth factor signaling with nutritional cues and synchronizes these upstream signals with the downstream stimulation of cell growth and proliferation (reviewed in reference 1). Akt activates mTORC1 in part by inhibiting the heterodimeric tuberous sclerosis complex (Tsc1/Tsc2). Tsc2 (or tuberin) functions as a GTPase-activating protein (GAP) to specifically inhibit the small GTPase Rheb, which activates mTORC1. The formation of a functional heterodimeric complex between Tsc2 and Tsc1 (or hamartin) is required for mTORC1 inhibition. As such, the disruption of the expression or function of either Tsc1 or Tsc2 is sufficient to activate mTORC1. Mammalian cells have evolved a negative feedback mechanism between mTORC1 and Akt to maintain an optimal balance between their activities. When Akt activates mTORC1, it initiates a negative feedback loop that serves to attenuate Akt activity. As such, mTORC1 serves as both an upstream and a downstream effector of Akt signaling. The loss of a functional Tsc1/Tsc2 complex disrupts this delicate balance, resulting in mTORC1 hyperactivity, which greatly reduces Akt activation (reviewed in reference 1). This is relevant to the heritable development of tuberous sclerosis in humans, which is caused by the mutational inactivation of either the TSC1 or TSC2 gene, leading to benign hamartoma formation and growth in a variety of organs (11).It is widely appreciated that low basal Akt activity renders Tsc1/2-deficient cells more sensitive to proapoptotic stimuli (4, 19). Unexpectedly, however, we found that both Tsc1 and Tsc2 null cells exhibit increased apoptotic resistance to growth factor withdrawal despite greatly reduced Akt activity relative to that of their wild-type counterparts. This implies that Tsc1/2 deficiency promotes or unmasks potent antiapoptotic mechanisms that reduce mammalian cell dependence upon growth factors and Akt for survival. Further investigation has uncovered a critical role for mTORC1 in promoting cell survival in the absence of growth factors.Trophic growth factors found in serum play a pivotal role in the cellular uptake and utilization of glucose, and serum withdrawal results in attenuated glucose metabolism. The maintenance of glucose utilization by the overexpression of the rate-limiting glycolytic enzyme hexokinase and its functionally coupled facilitative glucose transporters maintains cell survival in the absence of growth factors (reviewed in reference 18). We found that serum deprivation markedly increased both hexokinase II (HKII) and GLUT1 abundance in Tsc2-deficient cells, and the knockdown of HKII and GLUT1 increased the apoptotic susceptibility of these cells to serum deprivation. The elevated expression of HKII and GLUT1 is mediated by hypoxia-inducible factor 1α (HIF1α) protein, which is markedly induced by mTORC1 in serum-deprived Tsc2^−/− cells.In addition to increased HKII and GLUT1 expression, Tsc2^−/− cells display the sustained inhibition of glycogen synthase kinase 3 (GSK3) activity and stable Mcl-1 abundance following serum withdrawal, which also contribute to their apoptotic resistance under these conditions. Mcl-1 abundance, which normally declines following serum deprivation, is sustained in Tsc2^−/− cells by the constitutive inhibition of GSK3 and the activation of eIF4E.     

Contribution Ratios of amyA, amyB, amyC Genes to High-Level α-Amylase Expression in Aspergillus oryzae

Aspergillus oryzae strains express α-amylases abundantly, and the genome reference strain RIB40 has three α-amylase genes (amyA, amyB, and amyC). However, there is no information on the contribution ratios of individual α-amylase genes to total expression. In this study, we generated single, double, and triple disruptants of α-amylase genes by employing a strain (ΔligD) with high gene-targeting efficiency and pyrG marker recycling in A. oryzae. All the disruptants showed reduced activities of α-amylases, and the triple disruptant completely lost activity. Comparative analyses of the activities and mRNA amounts of the α-amylases suggest that the contribution of amyA to the α-amylase expression is smaller than those of amyB and amyC. The present study suggests that the ability to express a large amount of α-amylases in A. oryzae is attributed to gene duplication of genes such as amyB and amyC.     

Expression of connexin 37, 40, and 43 mRNA and protein in renal preglomerular arterioles

Gap junctions allow direct intercellular coupling between many cells including those in the vascular wall. Studies of connexin expression in cells of the microcirculatory system are very few in number. However, cell-to-cell communication between cells of the arteriolar wall may be particularly important in microcirculatory control. We investigated the expression of connexins 43, 40, and 37 (Cx43, Cx40, Cx37) mRNA and proteins in primary cultures of smooth muscle cells (SMC) from rat renal preglomerular arterioles and in the aortic cell line A7r5. Furthermore protein expression in preglomerular arterioles in frozen sections was evaluated. SMC were isolated from kidneys using an iron oxide sieve method and explant technique. Total RNA from these cultures was tested by RT-PCR analysis for the expression of the three connexins mRNA. Using immunofluorescence we examined whether the expression pattern of connexin protein in the cell culture and frozen sections corresponded to the mRNA expression. The data show that A7r5 and preglomerular SMC express mRNA for Cx37 in addition to Cx43 and Cx40. In A7r5 cells the mRNA for Cx43, Cx40, and Cx37 are translated to protein, whereas cultured preglomerular SMC and the media of afferent arterioles in frozen sections only showed Cx40 immunoreactivity.     

Expression, Purification and Characterization of a Recombinant β-Glucosidase from Volvariella Volvacea

For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a K_m of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside.