Pyruvate Decarboxylase of Zymomonas mobilis: Isolation, Properties, and Genetic Expression in Escherichia coli

Pyruvate decarboxylase (EC 4.1.1.1) from Zymomonas mobilis was purified to homogeneity by using dye-ligand and ion-exchange chromatography. Antibodies produced against the enzyme and the aminoterminalsequence obtained for the pure enzyme were used to select and confirm the identity of a genomic clone encoding the enzyme selected from a genomic library of Z. mobills DNA doned into pUC9. The genomic fragment encoding the enzyme expressed high levels of pyruvate decarboxylase in Escherichia coli. Possible RNA polymerase and ribosome-binding sites have been identified in the 5'-untranslated region of the pyruvate decarboxylase gene.

Cloning, expression and purification of the general stress protein yhbo fromEscherichia 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.

Predicting protein function from protein/protein interaction data: a probabilistic approach

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.

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.

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.

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.

Concerted Binding and Bending of DNA by Eschericia coli Integration Host Factor

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.