His-tagged Recombinant Protein Research Articles

Differential cold–adaptation among protein components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125

Thioredoxin and thioredoxin reductase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis were obtained as recombinant His-tagged proteins (rPhTrx and rPhTrxR, respectively). rPhTrxR is organised as a homodimeric flavoenzyme, whereas rPhTrx is a small monomeric protein, both containing a functional disulfide bridge. However, three additional cysteines are present as free thiols in purified rPhTrxR. When individually tested in specific assays, rPhTrxR and rPhTrx display a full activity at low temperatures, an indispensable requirement for cold-adapted proteins. In particular, rPhTrxR catalyses the NADPH dependent reduction of DTNB and rPhTrx provokes the insulin precipitation in the presence of DTT. The analysis of the effect of temperature on these reactions indicates that rPhTrxR is more cold-adapted than rPhTrx, having a higher psychrophilicity. The combined activity of rPhTrxR and rPhTrx, tested in a reconstituted assay containing NADPH as electrons donor and human insulin as the thioredoxin substrate, demonstrates a direct functional interaction between the purified recombinant components of the thioredoxin system of P. haloplanktis. Furthermore, the NADPH-dependent reduction of rPhTrx catalysed by rPhTrxR is fully reversible and allows the determination of its redox potential, whose value is in the range of other bacterial and archaeal thioredoxins. The analysis of the thermostability of rPhTrxR points to its discrete heat resistance. However, rPhTrx is much more heat resistant, with a half inactivation time of about 4 h at 95 degrees C. This exceptional heat resistance for a psychrophilic protein is significantly decreased by the reduction of the disulfide bridge of rPhTrx. Functionality, thermodependence and thermostability of the P. haloplanktis thioredoxin system point to the relevance of this key mechanism for the preservation of the reduced state of cytoplasmic proteins even in a cold-adapted source.

Functional expression of Francisella tularensis FabH and FabI, potential antibacterial targets

Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 μM with IC 50 value of 2 μM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 μM with IC 50 value of 6 μM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.

The d -2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis

Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding L-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (V(max)/K(m) ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted d-2-hydroxyacids but not l-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the d-mandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of D-2-hydroxyacid dehydrogenases which is unrelated to the well-described D-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD(+) necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.

Deactivation of Sphingosine Kinase 1 by Protein Phosphatase 2A

Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser(225), but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser(225). The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser(225) phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser(225) region of SK1. Finally, both basal and tumor necrosis factor-alpha-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation.

On the synthesis and rebinding properties of [Co(C 2H 3O 2) 2(z-Histidine)] imprinted polymers prepared by precipitation polymerization

In the present study, the synthesis via free-radical precipitation polymerization of metal-complexing polymer microparticles capable of specific binding to [Co(C 2H 3O 2) 2(z-Histidine)] complex is reported. Initially, the functional monomer (i.e., 4-vinylpyridine, a vinyl monomer with metal chelate properties) is complexed together with the template [Co(C 2H 3O 2) 2(z-Histidine)] molecule via the mediation of cobalt ions. Evidence of the polymerizable complex formation is obtained through the compleximetric titration of the z-Histidine in the prepolymerization reaction mixture. Subsequent polymerization of the polymerizable complex with a bi-functional or tri-functional cross-linker results in the formation of imprinted polymer microparticles in the size range of 1–5 µm. The effects of agitation, cross-linker type, degree of cross-linking and molar ratio of the template to the functional monomer on the rebinding properties of the imprinted polymers are experimentally assessed. The synthesized imprinted polymers can potentially find application in the purification of his-tagged recombinant proteins.

Turkey Coronavirus Non-Structure Protein NSP15 – An Endoribonuclease

Turkey coronavirus (TCoV) polyprotein was predicted to be cleaved into 15 non-structural proteins (nsp2 to nsp16), but none of these nsps have been characterized. TCoV nsp15 consists of 338 residues and shares 40% sequence similarity to U-specific Nidovirales endoribonuclease (NendoU) of severe acute respiratory syndrome coronavirus. Objective: The purpose of the present study was to characterize TCoV nsp15. Methods: The TCoV nsp15 gene was cloned into pTriEX1 and expressed as a C-terminal His-tagged recombinant protein in BL21 (DE3). The recombinant nsp15 was purified by Ni-NTA resin. Synthetic RNA substrates were used to determine the substrate specificity of the TCoV nsp15. RNA zymography was used to determine the active form of the nsp15. Results: The TCoV nsp15 did not cleave DNA but degraded total cellular RNA. The TCoV nsp15 cleaved single-stranded (ss) RNA at the uridylate site. The TCoV nsp15 cleaved hairpin RNA, pRNA, and double-stranded RNA (dsRNA) of infectious bursal disease virus very slowly, implying that dsRNA is not a good substrate for the TCoV nsp15. No divalent metal ion was required for in vitro enzymatic activity of the TCoV nsp15. The active form of the TCoV nsp15 was a homohexamer and disulfide bond was essential for the enzymatic activity. Conclusion: The TCoV nsp15 is a NendoU but has some characteristics different from other NendoU.

Enhancement of ABL Kinase Catalytic Efficiency by a Direct Binding Regulator Is Independent of Other Regulatory Mechanisms

ABL family tyrosine kinases are tightly regulated by autoinhibition and phosphorylation mechanisms. These kinases maintain an inactive conformation through intramolecular interactions involving SH3 and SH2 domains. RIN1, a downstream effector of RAS, binds to the ABL SH3 and SH2 domains and stimulates ABL tyrosine kinase activity. RIN1 binding to the ABL2 kinase resulted in a large decrease in Km and a small increase in Vmax toward an ABL consensus substrate peptide. The enzyme efficiency (k(cat)/Km) was increased more than 5-fold by RIN1. In addition, RIN1 strongly enhanced ABL-mediated phosphorylation of CRK, PSTPIP1, and DOK1, all established ABL substrates but with unique protein structures and distinct target sequences. Importantly RIN1-mediated stimulation of ABL kinase activity was independent of activation by SRC-mediated phosphorylation. RIN1 increased the kinase activity of both ABL1 and ABL2, and this occurred in the presence or absence of ABL regulatory domains outside the SH3-SH2-tyrosine kinase domain core. We further demonstrate that a catalytic site mutation associated with broad drug resistance, ABL1T315I, remains responsive to stimulation by RIN1. These findings are consistent with an allosteric kinase activation mechanism by which RIN1 binding promotes a more accessible ABL catalytic site through relief of autoinhibition. Direct disruption of RIN1 binding may therefore be a useful strategy to suppress the activity of normal and oncogenic ABL, including inhibitor-resistant mutants that confound current therapeutic strategies. Stimulation through derepression may be applicable to many other tyrosine kinases autoinhibited by coupled SH3 and SH2 domains.

Arabidopsis ACBP6 is an acyl-CoA-binding protein associated with phospholipid metabolism

In our recent paper in Plant Physiology, we showed that the Arabidopsis thaliana 10-kD acyl-CoA-binding protein, ACBP6, is subcellularly localized to the cytosol and that the overexpression of ACBP6 in transgenic Arabidopsis enhanced freezing tolerance. ACBP6-conferred freezing tolerance was independent of induced cold-regulated (COLD-RESPONSIVE) gene expression, but was correlated to an enhanced expression of phospholipase Dδ (PLDδ). Lipid analyses on cold-acclimated freezing-treated ACBP6-overexpressors revealed a decline in phosphatidylcholine (PC) and an elevation of phosphatidic acid (PA) in comparison to wild type. Furthermore, the His-tagged ACBP6 recombinant protein was observed using in vitro filter-binding assays to bind PC, but not PA or lysophosphatidylcholine. Taken together, our results implicate roles for ACBP6 in phospholipid metabolism that is related to gene regulation and PC-binding/transfer. This represents the first report demonstrating the in vitro binding of an ACBP to a phospholipid. The effect of ACBP6 on PLDδ expression is reminiscent of yeast 10-kD ACBP function in the regulation of genes associated with stress responses, fatty acid synthesis and phospholipid synthesis. However, the yeast ACBP regulates the expression of genes involved in phospholipid synthesis by donation of acyl-CoA esters and its binding to phospholipids remains to be demonstrated.Addendum to: Chen Q-F, Xiao S, Chye M-L. Overexpression of the Arabidopsis 10-Kilodalton acyl-CoA-binding protein, ACBP6, enhances freezing tolerance. Plant Physiol 2008; dio:10.1104/pp.108.123331

Vibrio vulnificus rpoS Expression Is Repressed by Direct Binding of cAMP-cAMP Receptor Protein Complex to Its Two Promoter Regions

Vibrio vulnificus, a septicemia-causing pathogenic bacterium, acquires resistance against various stresses and expresses virulence factors via an rpoS gene product. In this study, we investigated the transcriptional characteristics of this global regulator. Two distinct transcriptional initiation sites for the rpoS gene, the proximal promoter (P(p)) and the distal promoter (P(d)), were defined by primer extension experiments. Various rpoS::luxAB transcriptional fusions indicated that P(d) is a major promoter of rpoS expression. Western blot analysis showed that RpoS levels were inversely correlated with intracellular levels of 3',5'-cyclic AMP (cAMP). The expressions of both P(d) and P(p) were increased in cya and crp mutants. The exogenous addition of cAMP to the cya mutant resulted in repressed expression of rpoS. In addition, rpoS expression was significantly lowered in the cpdA mutant, in which the level of cAMP was elevated because of the absence of 3',5'-cAMP phosphodiesterase. In vitro transcription assays using the V. vulnificus RNA polymerase showed that the transcripts from both promoters were reduced by addition of the cAMP-cAMP receptor protein (CRP). The cAMP-CRP was shown to bind to two rpoS promoters by electrophoretic mobility shift assays. The alteration of the putative CRP-binding site on each rpoS promoter, via site-directed mutagenesis, abolished the binding of cAMP-CRP as well as regulation by cAMP-CRP. Therefore, this study shows a relationship between the level of intracellular cAMP and the degree of rpoS expression and further demonstrates, for the first time, the direct binding of the cAMP-CRP complex to rpoS upstream regions, which results in repression of rpoS gene expression.

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added. Then, bilayers are charged with Ni and his-tagged recombinant proteins are added. Finally, lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting, and sites of receptor degradation.

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added. Then, bilayers are charged with Ni and his-tagged recombinant proteins are added. Finally, lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting, and sites of receptor degradation.

The phage K lytic enzyme LysK and lysostaphin act synergistically to kill MRSA

LysK is the endolysin from the staphylococcal bacteriophage K, and can digest the cell wall of many staphylococci. Lysostaphin is a bacteriocin secreted by Staphylococcus simulans to kill Staphylococcus aureus. Both LysK and lysostaphin have been shown to lyse methicillin-resistant S. aureus (MRSA). This study describes optimal reaction conditions for the recombinant His-tagged LysK protein (pH range pH 6-10, and 0.3-0.5 M NaCl), and C-His-LysK MIC (32.85+/-4.87 mug mL(-1)). LysK and lysostaphin demonstrate antimicrobial synergy by the checkerboard assay.

Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKɛ-mediated IRF activation

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through IKKepsilon and TBK1 and consequently IFN-beta induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-beta induction by preventing TBK1/IKKepsilon-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKepsilon, whereas knockdown of DDX3 inhibited this, and virus- or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA- and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKepsilon dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKepsilon. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKepsilon-mediated IRF activation and Ifnb promoter induction.

Induction of Shrimp Tropomyosin-Specific Hypersensitivity in Mice

Background: Shellfish hypersensitivity is amongst the most common food allergies. The major shellfish allergen was identified as tropomyosin. Here, we investigated the immediate hypersensitivity responses, IgE and cell-mediated immune response in mice sensitized with recombinant shrimp tropomyosin. Methods: Shrimp tropomyosin was cloned and expressed as a His-tagged fusion recombinant protein in Escherichia coli. Three- to 4-week-old BALB/c mice were sensitized by intragastric administration of recombinant tropomyosin (0.1 mg) plus cholera toxin (10 µg) on days 0, 12, 19 and 26 and challenged on day 33. Mice fed with phosphate-buffered saline plus cholera toxin were included as controls. Animals were monitored for immediate hypersensitive responses and tropomyosin-specific IgE over time. In addition, shrimp tropomyosin-specific CD4+ T cells, interleukin-4 and interferon-γ levels were determined from in vitro splenocyte cultures. A passive cutaneous anaphylaxis assay was also conducted. Results: Mice fed with shrimp tropomyosin developed swelling of the snout, increased scratching behavior and shrimp tropomyosin-specific IgE. Sera from tropomyosin-sensitized mice elicited vascular leakage in naïve mice in the passive cutaneous anaphylaxis assay. Shrimp tropomyosin-specific CD4+ T cell proliferations and elevated interleukin-4 over interferon-γ levels were evident in splenocyte cultures of tropomyosin-fed mice upon tropomyosin stimulation. In contrast, shrimp tropomyosin-specific IgE, CD4+ T cells and hypersensitive responses were absent in the control mice. Conclusion: We have generated a BALB/c model of shrimp allergy. This model provides a useful tool for evaluating the immunopathogenic mechanisms involved in shellfish hypersensitivity.

Screening of a HUVEC cDNA library with transplant-associated coronary artery disease sera identifies RPL7 as a candidate autoantigen associated with this disease.

A HUVEC cDNA library was screened with sera from two patients who had developed transplant-associated coronary artery disease (TxCAD) following cardiac transplantation. A total of six positive clones were isolated from a primary screen of 40 000 genes. Subsequent DNA sequence analysis identified these to be lysyl tRNA synthetase, ribosomal protein L7, ribosomal protein L9, beta transducin and TANK. Another gene whose product could not be identified showed homology to a human cDNA clone (DKFZp566M063) derived from fetal kidney. Full-length constructs of selected genes were expressed as his-tag recombinant fusion proteins and used to screen a wider patient base by ELISA to determine prevalence and association with TxCAD. Of these ribosomal protein L7 showed the highest prevalence (55.6%) with TxCAD sera compared to 10% non-CAD.

Interaction and Stoichiometry of the Peripheral Stalk Subunits NtpE and NtpF and the N-terminal Hydrophilic Domain of NtpI of Enterococcus hirae V-ATPase

The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.

Overexpression, Refolding, Purification of Erbin PDZ Domain from Escherichia coli and Preparation of its Polyclonal Antibody

The Erbin was recently identified. The antibody against Erbin has not been commercially available. As a new member of peripheral protein LAP family and novel type of adaptor protein, its functions and binding partners are not completely known. In the present study, cDNA encoding PDZ domain of Erbin was inserted in a prokaryotic expression vector. His-tagged recombinant protein was overproduced in E. coli and purified by Ni-NTA column chromatography. About 14.4 mg of the purified protein was obtained from 500 mL of cell culture. The purity of the recombinant protein was higher than 90%. The polyclonal antibody against this protein was raised. The antibody can recognize both denatured and natural Erbin protein. It will be used to further identify the new binding partners of Erbin and study its unknown functions.

Decreased Ceramide Transport Protein (CERT) Function Alters Sphingomyelin Production following UVB Irradiation

Increased cellular ceramide accounts in part for UVB irradiation-induced apoptosis in cultured human keratinocytes with concurrent increased glucosylceramide but not sphingomyelin generation in these cells. Given that conversion of ceramide to non-apoptotic metabolites such as sphingomyelin and glucosylceramide protects cells from ceramide-induced apoptosis, we hypothesized that failed up-regulation of sphingomyelin generation contributes to ceramide accumulation following UVB irradiation. Because both sphingomyelin synthase and glucosylceramide synthase activities were significantly decreased in UVB-irradiated keratinocytes, we investigated whether alteration(s) in the function of ceramide transport protein (or CERT) required for sphingomyelin synthesis occur(s) in UVB-irradiated cells. Fluorescently labeled N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-ceramide) relocation to the Golgi was diminished after irradiation, consistent with decreased CERT function, whereas the CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (1R,3R isomer) (HPA-12) produced an equivalent effect. UVB irradiation also induced the rapid formation of a stable CERT homotrimer complex in keratinocytes as determined by Western immunoblot and mass spectrometry analyses, a finding replicated in HeLa, HEK293T, and HaCaT cells and in murine epidermis. Ceramide binding activity was decreased in recombinant CERT proteins containing the UVB-induced homotrimer. The middle region domain of the CERT protein was required for the homotrimer formation, whereas neither the pleckstrin homology (Golgi-binding) nor the START (ceramide-binding) domains were involved. Finally like UVB-treated keratinocytes, HPA-12 blockade of CERT function increased keratinocyte apoptosis, decreased sphingomyelin synthesis, and led to accumulation of ceramide. Thus, UVB-induced CERT homotrimer formation accounts, at least in part, for apoptosis and failed up-regulation of sphingomyelin synthesis following UVB irradiation, revealing that inactive CERT can attenuate a key metabolic protective mechanism against ceramide-induced apoptosis in keratinocytes.

Substrate Specificity of the 3-Methylcrotonyl Coenzyme A (CoA) and Geranyl-CoA Carboxylases from Pseudomonas aeruginosa

Biotin-containing 3-methylcrotonyl coenzyme A (MC-CoA) carboxylase (MCCase) and geranyl-CoA (G-CoA) carboxylase (GCCase) from Pseudomonas aeruginosa were expressed as His-tagged recombinant proteins in Escherichia coli. Both native and recombinant MCCase and GCCase showed pH and temperature optima of 8.5 and 37 degrees C. The apparent K(0.5) (affinity constant for non-Michaelis-Menten kinetics behavior) values of MCCase for MC-CoA, ATP, and bicarbonate were 9.8 microM, 13 microM, and 0.8 microM, respectively. MCCase activity showed sigmoidal kinetics for all the substrates and did not carboxylate G-CoA. In contrast, GCCase catalyzed the carboxylation of both G-CoA and MC-CoA. GCCase also showed sigmoidal kinetic behavior for G-CoA and bicarbonate but showed Michaelis-Menten kinetics for MC-CoA and the cosubstrate ATP. The apparent K(0.5) values of GCCase were 8.8 microM and 1.2 microM for G-CoA and bicarbonate, respectively, and the apparent K(m) values of GCCase were 10 microM for ATP and 14 microM for MC-CoA. The catalytic efficiencies of GCCase for G-CoA and MC-CoA were 56 and 22, respectively, indicating that G-CoA is preferred over MC-CoA as a substrate. The enzymatic properties of GCCase suggest that it may substitute for MCCase in leucine catabolism and that both the MCCase and GCCase enzymes play important roles in the leucine and acyclic terpene catabolic pathways.

Multifunctional polymer-derivatized γ-Fe 2O 3 nanocrystals as a methodology for the biomagnetic separation of recombinant His-tagged proteins

Multifunctional polymer-derivatized superparamagnetic iron oxide (γ-Fe 2O 3) nanoparticles were prepared for biomagnetic separation of histidine-tagged recombinant proteins building up a faster and efficient method for protein separation by making use of their intrinsic magnetic properties. Using polymer bound γ-Fe 2O 3 nanocrystals, a 6× histidine-tagged recombinant protein (silicatein) with a molecular weight of 24 kDa has been isolated and purified. The supermagnetic iron oxide nanocrystals were characterized by transmission electron microscopy (TEM), high-resolution TEM (HRTEM), SQUID and Mössbauer and the polymer functionalization of the γ-Fe 2O 3 nanocrystals was monitored by UV–vis spectroscopy and light microscopy. Protein immobilization and separation was monitored using immunostaining techniques and gel electrophoresis, respectively.