Predicting protein function from protein/protein interaction data

Motivation: The development of experimental methods for genome scale analysis of molecular interaction networks has made possible new approaches to inferring protein function. This paper describes a method of assigning functions based on a probabilistic analysis of graph neighborhoods in a protein-protein interaction network. The method exploits the fact that graph neighbors are more likely to share functions than nodes which are not neighbors. A binomial model of local neighbor function labeling probability is combined with a Markov random field propagation algorithm to assign function probabilities for proteins in the network. Results: We applied the method to a protein-protein interaction dataset for the yeast Saccharomyces cerevisiae using the Gene Ontology (GO) terms as function labels. The method reconstructed known GO term assignments with high precision, and produced putative GO assignments to 320 proteins that currently lack GO annotation, which represents about 10% of the unlabeled proteins in S. cerevisiae.

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High-level Expression of Cecropin X in Escherichia coli

Cecropin X is a short cationic peptide with a broad antibacterial and antitumor spectrum. Here, we report the production of a tumor necrosis factor (TNFα)-cecropin X fusion protein under the control of a temperature-inducible PR promoter in the bacterial expression vector pRC. During fermentation, we studied and optimized essential parameters including the type of host cells, medium, timing of induction, post-induction time and dissolved oxygen level. Using the suitable conditions in the fermentation, up to 20 % - 23 % of the total cellular proteins is produced as the fusion protein, mostly in the form of inclusion bodies. After washing, on average about 5.27 g dried inclusion bodies could be collected from 1 L broth and the purity of inclusion bodies reached 80 %. Cecropin X obtained by cleaving the fusion protein with cyanogen bromide showed remarkable tumorcidal activity against mouse Lewis lung carcinoma 3LL in vivo.

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High efficiency transformation of E.coli by high voltage electroporation

E. coil can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 109 to 1010 transformants/jg with strains LE392 and DH5ox, and plasmids pUC18 and pBR329. The process is highly dependent on two characterstics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.

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Concerted Binding and Bending of DNA

Integration host factor (IHF) is a heterodimeric Eschericia coli protein that plays essential roles in a variety of cellular processes including site specific recombination, transcription, and DNA replication. The IHFDNA interface extends over three helical turns and includes sequential minor groove contacts that present strong, sequence specific protection patterns against hydroxyl radical cleavage. Synchrotron X-ray footprinting has been used to follow the kinetics of formation of DNA-protein contacts in the IHF-DNA complex with single base-pair spatial, and millisecond time, resolution. The three sites of IHF protection on the DNA develop with similar time-dependence, indicating that sequence specific binding and bending occur concertedly. Two distinct phases are observed in the association process. The first ``burst'' phase is characterized by a rate that is greater than diffusion limited (>1010 sÿ1 Mÿ1) and the second phase is on the order of diffusion controlled (_108 Mÿ1 sÿ1). The overall kinetics of association become faster with increasing IHF concentration showing that complex formation is second-order with protein. The rate of association is maximal between 100 and 200 mM KCl decreasing at higher and lower concentrations. The rate of IHF dissociation from site specifically bound DNA increases monotonically as KCl concentration is increased. The dissociation progress curves are biphasic with the amplitude of the first phase dependent upon competitor DNA concentration. These results are the first analysis by synchrotron footprinting of the fast kinetics of a protein-DNA interaction and suggest that IHF binds its specific site through a multiple-step mechanism in which the first step is facilitated diffusion along the length of the duplex followed by subsequent binding and bending of the DNA in a concerted manner.

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Cloning and Expression of Coxsakievirus B3 Viral Protein-1

Background: Viral protein-1 (VP1) is a major capsid protein of Coxsakievirus B3 (CVB3) that plays an important role in directing viruses towards permissive cells and acts as a main antigenic site of the virus in eliciting of host immune response, hence it seems VP1 can be considered as a vaccine candidate against CVB3 infection. In this study, cDNA of VP1 was prepared, cloned into pET expression vector and the recombinant protein (VP1) was over expressed in E. coli. Methods: The viruses were grown in suspension cultures of Vero cells with an input virus multiplicity of 10-50 plaque-forming units/cell. After observing complete cytopathic effect, the total RNA (cells and virus) was prepared for RT-PCR and by using specific primers, VP1 cDNA was amplified and ligated into pET vectors (32 a and 28 a). The recombinant vector was transferred into competent E. coli (BL-21) and after selection of proper colony, which carried correct cDNA within the vector; cells were cultured and induced with isopropyl B-D-thiogalactopyranoside, in order to express protein (VP1). The cultures were tested for presence of VP1 by SDS-PAGE and Western-Blotting analysis. Results: Molecular techniques such as PCR which showed exact defined size of the VP1 (819 bp), restriction digestion and finally immunoblot analysis of over expressed protein; all confirmed the correct cloning and expression of VP1 in this research. Conclusion: In this research, full length of VP1 as major capsid protein of CVB3 was over expressed in E. coli which, can be used for further studies, including neutralizing antibody production against CVB3.

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CLONING, EXPRESSION AND PURIFICATION OF THE GENERAL STRESS PROTEIN YhbO FROM Escherichia coli

We cloned, expressed and purified the Escherichia coli yhbO gene product, which is homolog to the Bacillus subtilis general stress protein 18 (the yfkM gene product), the Pyrococcus furiosus intracellular protease PfpI, and the human Parkinson disease protein DJ-1. The gene coding for YhbO was generated by amplifying the yhbO gene from E. coli by polymerase chain reaction. It was inserted in the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21(DE3) E. coli strain transformed with the YhbO-expression vector pET-21a-yhbO, accumulates large amounts of a soluble protein of 20 kDa in SDS-PAGE that matches the expected YhbO molecular weight. YhbO was purified to homogeneity by HPLC DEAE ion exchange chromatography and hydroxylapatite chromatography and its identity was confirmed by N-terminal sequencing and mass spectrometry analysis. The native protein exists in monomeric, trimeric and hexameric forms.

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Expression of green fluorescent protein (GFPuv) in Escherichia coli DH5-α, under different growth conditions

The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by a fractional factorial (24-1) design at two levels: (i) the effect of storing (24-48 h) the seeded broth at 4oC prior to incubation at 37oC; (ii) the effect of agitation speed (100-200 rpm); (iii) the final concentration (0.05-0.5 mM) of IPTG (isopropyl–β-D-thiogalactopyranoside) and (iv) the addition of IPTG at set cell densities (OD660 0.01-0.8). GFPuv was extracted from cells by the three phase partitioning method (TPP) and further purified with a methyl HIC column. The cultures grown at 37oC/24 h provided the highest yields of GFPuv under the conditions: (i) pre-storage at 4oC/24 h; (ii) agitation speed at 100 rpm; (iii) 0.5 mM IPTG and (iv) IPTG addition at OD660~0.01. On the other hand, at 37oC/ 8 h, GFPuv expression was dependent upon agitation of broth cultures at 200 rpm and the IPTG addition at the beginning of the growth exponential phase.

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Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene (pdc) and Comparison to Bacterial Homologues

Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial pdc genes have been previously described and expressed as active recombinant proteins. The pdc gene from Zymomonas mobilis has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial pdc gene from Zymobacter palmae. The pattern of codon usage for this gene appears quite similar to that for Escherichia coli genes. In E. coli recombinants, the Z. palmae PDC represented approximately 1/3 of the soluble protein.
Biochemical and kinetic properties of the Z. palmae enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the Z. palmae enzyme exhibited the highest specific activity (130 U mg of protein_1) and the lowest Km for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.


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Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paraná, Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO® vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.

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Genetic Engineering of Zymobacter palmae for Ethanol Production from Xylose

Its metabolic characteristics suggest Zymobacter palmae gen. Nov., sp. Nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation will require certain genetic modifications. We therefore engineered Z. palmae so as to broaden the range of its fermentable sugar substrates to include the pentose sugar xylose. The Escherichia coli genes encoding the xylose catabolic enzymes xylose isomerase (XI), xylulokinase (XK), transaldolase (TA) and transketolase (TK) were introduced into Zb. palmae, where their expression was driven
by the Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. When cultured with 40 g/l xylose, the recombinant Z. palmae was able to ferment 16.4 g/l xylose within 5 days, producing 91% of the theoretical yield of ethanol with no accumulation of organic acids as metabolic by-products. Notably, xylose-acclimation enhanced both the expression of xylose catabolic enzymes and the rate of xylose uptake into recombinant Z. palmae, which enabled the acclimated organism to completely and simultaneously ferment a mixture of 40 g/l glucose and 40 g/l xylose within 8 h, producing 95% of theoretical yield of ethanol. Thus, efficient fermentation of a mixture of glucose and xylose to ethanol can be accomplished using Z. palmae expressing E. coli xylose catabolic enzymes.

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