Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

The left ventricular contractility of the rat heart is modulated by changes in flow and a1-adrenoceptor stimulation

Myocardial contractility depends on several mechanisms such as coronary perfusion pressure (CPP) and flow as well as on a1-adrenoceptor stimulation. Both effects occur during the sympathetic stimulation mediated by norepinephrine. Norepinephrine increases force development in the heart and produces vasoconstriction increasing arterial pressure and, in turn, CPP. The contribution of each of these factors to the increase in myocardial performance needs to be clarified. Thus, in the present study we used two protocols: in the first we measured mean arterial pressure, left ventricular pressure and rate of rise of left ventricular pressure development in anesthetized rats (N = 10) submitted to phenylephrine (PE) stimulation before and after propranolol plus atropine treatment. These observations showed that in vivo a1-adrenergic stimulation increases left ventricular-developed pressure (P<0.05) together with arterial blood pressure (P<0.05). In the second protocol, we measured left ventricular isovolumic systolic pressure (ISP) and CPP in Langendorff constant flow-perfused hearts. The hearts (N = 7) were perfused with increasing flow rates under control conditions and PE or PE + nitroprusside (NP). Both CPP and ISP increased (P<0.01) as a function of flow. CPP changes were not affected by drug treatment but ISP increased (P<0.01). The largest ISP increase was obtained with PE + NP treatment (P<0.01). The results suggest that both mechanisms, i.e., direct stimulation of myocardial a1-adrenoceptors and increased flow, increased cardiac performance acting simultaneously and synergistically.

Wavelength Selective a-SiC:H p-i-n/p-i-n Heterostructure for Fluorescent Proteins Detection

In this paper we present results on the optimization of multilayered a-SiC:H heterostructures that can be used as optical transducers for fluorescent proteins detection using the Fluorescence Resonance Energy Transfer approach. Double structures composed by pin based aSiC:H cells are analyzed. The color discrimination is achieved by ac photocurrent measurement under different externally applied bias. Experimental data on spectral response analysis, current-voltage characteristics and color and transmission rate discrimination are reported. An electrical model, supported by a numerical simulation gives insight into the device operation. Results show that the optimized a-SiC:H heterostructures act as voltage controlled optical filters in the visible spectrum. When the applied voltages are chosen appropriately those optical transducers can detect not only the selective excitation of specimen fluorophores, but also the subsequent weak acceptor fluorescent channel emission.

The folding of knotted proteins: insights from lattice simulations

We carry out systematic Monte Carlo simulations of Go lattice proteins to investigate and compare the folding processes of two model proteins whose native structures differ from each other due to the presence of a trefoil knot located near the terminus of one of the protein chains. We show that the folding time of the knotted fold is larger than that of the unknotted protein and that this difference in folding time is particularly striking in the temperature region below the optimal folding temperature. Both proteins display similar folding transition temperatures, which is indicative of similar thermal stabilities. By using the folding probability reaction coordinate as an estimator of folding progression we have found out that the formation of the knot is mainly a late folding event in our shallow knot system.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Electrochemical studies on small electron transfer proteins using membrane electrodes

Journal of Electroanalytical Chemistry 541 (2003) 153-162

Structural and mechanistic studies of iron containing proteins

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica, ramo de Bioquímica-Física, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Molybdenum-containing proteins from Sulphate Reducing Bacteria: studying proteins with novel cofactors and revealing new features of old enzymes

Dissertação apresentada para a obtenção do Grau de Doutor em Bioquímica, especialidade de Bioquímica-Física pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Elution of proteins from polyacrylamide eels: a simple and economic procedure

A simplified methodology for the quantitative electroelution of proteins from polyacrylamide gels is described. After staining with Coomassie Brilliant Blue R 250, the identified bands are excised from the gel and the proteins eluted using a procedure developed for use in conventional tube gel electrophoresis equipment.

Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins

Glucose monitoring in vivo is a crucial issue for gaining new understanding of diabetes. Glucose binding protein (GBP) fused to two fluorescent indicator proteins (FLIP) was used in the present study such as FLIP-glu- 3.2 mM. Recombinant Escherichia coli whole-cells containing genetically encoded nanosensors as well as cell-free extracts were immobilized either on inner epidermis of onion bulb scale or on 96-well microtiter plates in the presence of glutaraldehyde. Glucose monitoring was carried out by Förster Resonance Energy Transfer (FRET) analysis due the cyano and yellow fluorescent proteins (ECFP and EYFP) immobilized in both these supports. The recovery of these immobilized FLIP nanosensors compared with the free whole-cells and cell-free extract was in the range of 50–90%. Moreover, the data revealed that these FLIP nanosensors can be immobilized in such solid supports with retention of their biological activity. Glucose assay was devised by FRET analysis by using these nanosensors in real samples which detected glucose in the linear range of 0–24 mM with a limit of detection of 0.11 mM glucose. On the other hand, storage and operational stability studies revealed that they are very stable and can be re-used several times (i.e. at least 20 times) without any significant loss of FRET signal. To author's knowledge, this is the first report on the use of such immobilization supports for whole-cells and cell-free extract containing FLIP nanosensor for glucose assay. On the other hand, this is a novel and cheap high throughput method for glucose assay.

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability

Journal of Proteome Research (2006)5: 2720-2726

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins

Journal of Biological Inorganic Chemistry (2010)15: 271-281

Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS

Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-g by lymphocyte from subjects vaccinated with Leishvacinâ . The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mg of each protein at 15 days interval. One hundred mg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60) and 10.77UI/ml of IFN-g production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis.