Construction, expression, purification and functional analysis of recombinant NF{kappa}B p50/p65 heterodimer

NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B plays an important role in mediating the gene expressionof numerous cellular processes such as growth, development,the inflammatory response and virus proliferation. The p50/p65heterodimer is the most abundant form of the NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B dimers andplays a more elaborate role in gene regulation. Biochemicalresearch on p50/p65 NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B has not benefited however from the availabilityof easily purified recombinant protein. We report two methodsfor the large scale expression and purification of recombinantNF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B p50/p65 heterodimer. The first utilizes a bacterial doubleexpression vector which contains two ribosomal binding sitesto facilitate the coexpression of the polypeptides in the p50/p65NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B heterodimer. The second method uses a mixed protein refoldingstrategy. Both methods yield crystallizable protein. Electrophoreticmobility shift assays confirm that the DNA binding affinityis independent of the method used to purify the protein. Thesemethods will facilitate the numerous studies on various NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B/Relfamily members.     

Effects of novel peptides derived from the acidic tail of synuclein (ATS) on the aggregation and stability of fusion proteins

The acidic tail of alpha-synuclein (ATSalpha) has been shown to protect the glutathione S-transferase (GST)-ATSalpha fusion protein from environmental stresses, such as heat, pH and metal ions. In this study, we further demonstrated that the introduction of ATSalpha into other proteins, such as dehydrofolate reductase and adiponectin, renders the fusion proteins resistant to heat-induced aggregation and that the acidic tail of beta- or gamma-synuclein can also protect the fusion proteins from heat-induced aggregation. Interestingly, the heat resistance of GST-ATSalpha deletion mutants, which contain shorter peptides derived from the highly charged regions of ATSalpha, was approximately proportional to the number of added Glu/Asp residues. However, the negative charges in the ATSalpha-derived peptides appear insufficient to explain the extreme heat resistance of the fusion proteins, since polyglutamates appeared to be much less effective than the ATSalpha-derived peptides in conferring heat resistance on the fusion proteins. These results suggest that not only the negatively charged residues but also the specific amino acid sequence of ATSalpha play an important role in conferring extreme heat resistance on the fusion proteins. Furthermore, the heat-induced secondary structural changes and thermal inactivation curves of GST-ATSalpha deletion mutants indicated that the introduction of ATSalpha-derived peptides does not significantly affect the intrinsic stability of the fusion proteins.     

On Non-axiomatizability of Superintuitionistic Predicate Logics of Some Classes of Well-founded and Dually Well-founded Kripke Frames

The article presents general results on non-axiomatizabilityfor superintuitionistic predicate logics. In particular, thelogics of all well-ordered, all dually well-ordered, and alldually well-founded Kripke frames (in the semantics with nestedand with constant domains) are <IMG BORDER=0 WIDTH=13 HEIGHT=15 SRC="http://logcom.oxfordjournals.org/content/vol16/issue5/images/medium/exl031i34.gif" ALT="Formula " ALIGN="bottom"> -hard, and the logic of all Kripke frames of finite heightis not recursively axiomatizable (although it is known to be<IMG BORDER=0 WIDTH=14 HEIGHT=14 SRC="http://logcom.oxfordjournals.org/content/vol16/issue5/images/medium/exl031i33.gif" ALT="Formula " ALIGN="bottom"> -arithmetical). A resulton Kripke-incompleteness is stated as well.     

Structure-function validation of high lysine analogs of {alpha}-hordothionin designed by protein modeling

Cereal grains and legume seeds, which are key protien sourcesfor the vegetarian diet, are generally deficient in essentialamino acids. Maize, in particular, is deficient in lysine. Theinherent lack of lysine-rich protiens in maize has necessitatedthe search for heterologous protiens enriched in this aminoacid, the isolation of the corresponding gene and its ultimateintroduction into maize through plant transformation techniques.However, a rate-limiting step to this strategy has been theavailability of plant-derived lysine-rich protiens. An appealingsolution to the problem is to artificially increase the lysinecontent of a given protein by mutating appropriate residuesto lysine. Here, we expound this strategy, starting with theprotein <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> hordothionin that is derived from barley seeds andconsists of five lysine residues in a total of 45 amino acids(11 % lysine). To facilitate rational substitutions, the 3-Dstructure of the protein has been determined by homology modelingwith crambin. based on this model, we have identified surfaceresidues amenable to substitution with lysine. Furthermore,the acceptability of the mutations has been validated throughthe syntheses and characterization of the derivatives. To thisend, our approach has permitted the creation of a modified <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-hordothionin protein that has a lysine content of 27 % andretains the antifungal activity of the wild-type protein.     

The prediction and orientation of {alpha}-helices from sequence alignments: the combined use of environment-dependent substitution tables, Fourier transform methods and helix capping rules

Amino acid substitution tables are used to estimate the extentto which amino acids in families of homologous proteins areexposed to the solvent. The approach depends on the comparisonof difference environment-dependent tables for solvent accessible/inaccessibleresidues with amino acid substitutions at each position in analigned set of sequences. The periodicity in the predicted accessible/inaccessibleresidues is calculated using a Fourier transform procedure modifiedfrom that used to calculate hydrophobic moments. a-Helices areidentified from the characteristic periodicities and the solventaccessible face of the helix is defined. The initial helix predictionsare refined using rules for identifying the N- and C-terminiof helices from sequence alignments. These rules have been definedfrom a study of protein structures. The combined method correctlypredicts 79% of the residues in helices and incorrectly predictsonly 12% of the nonhelical residues as helical. In addition,since the method is reliable at predicting the correct numberof helices in the correct position in the sequence and sinceit also predicts the internal face of each helix, the resultscan be used to postulate 3-D arrangements of the secondary structureelements.     

Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations

The structure of endothelin-1 (ET-1), an endothelial cell-derivedpeptide with vasoconstricting activity, was determined in anaqueous solution by means of a combination of NMR and distancegeometry calculations. The resulting structure is characterizedby an <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helical conformation in the sequence region, Lys9-Cys15.Furthermore, an extended structure and a turn structure existin the Cys1-Ser4 and Ser5-Asp8 regions respectively, and nopreferred conformation was found for the C-terminal part ofthe peptide which was not uniquely constrained by the NMR data.These structural elements, the <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helical structure in the sequenceportion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys,are homologous to those found commonly in several neurotoxicpeptides.     

The solution structure and conformational dynamics of tumor necrosis factor-{alpha} and a (Cys69 - Asp; Cys101 - Arg) analog as examined by IR spectroscopy and hydrogen exchange

An analog of human tumor necrosis factor-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> (TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">) was createdwith Cys69 and CyslOl replaced with Asp and Arg respectively.We have undertaken a comparative study of the solution conformationand dynamics of the native and analog molecules using a combinationof Fourier transform IR spectroscopy and hydrogen-deuterium(H-D) exchange kinetics. IR spectroscopic results indicate thatthe analog molecule adopts a gross structure similar to thatof the native molecule but significant differences in the conformationof the ß-sheets are observed. Increased bandwidthsobserved for several of the amide I components also suggesta less rigid structure for the analog molecule. Further, bymonitoring the frequency shifts of the individual amide I componentbands as a function of hydrogen exchange, we have enhanced ourability to assign these components to individual protein secondarystructures, particularly the high frequency ß-strandmode. Hydrogen exchange kinetic studies indicate that the Asp–Arganalog adopts a looser, more flexible solution structure relativeto the natural sequence molecule.     

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the {alpha}/{beta}-barrel Proteins

We have attempted to construct an artificial polypeptide thatfolds like the eight-stranded parallel ß-barrel structures.Our approach consists of repeating eight times a unit peptidedesigned to adopt a ‘ß-strand/<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helix’pattern. A first ‘test’ sequence for this structuralunit was deduced from a series of parameters defined after ananalysis of three natural <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">/ß-barrel proteins and includingprincipally the lengths of the secondary structure elements,the <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">/ß packing and the fitting on average Garnierprofiles. The gene encoding this structural unit was synthesized,cloned and expressed in Escherichia coli either as a monomeror as direct repeats of 2–12 units. Preliminary structuralcharacterization of the 7-, 8- and 9-fold unit polypeptidesby circular dichroism measurements indicates the presence ofthe predicted amount of <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helix in the three proteins. Furtheranalysis by urea-gradient gel electrophoresis demonstrates that,in the conditions tested, only the 8-fold unit polypeptide formsa compact structure through a cooperative and rapid two-statefolding transition involving long-range molecular interactions.     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor {alpha}

A gene encoding human tumour necrosis factor <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> (TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">) has beenchemically synthesized, cloned and expressed to yield a biologicallyactive protein in Escherichia coli. The 480-bp gene was assembledby enzymic ligation of 32 oligonucleotides, cloned directlyinto M13mp18 for sequence verification and expressed in thebroad host range high-level expression vector pMMB66EHST. Expressedrecombinant TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> was shown to have the correct molecular weight,processed N-terminal sequence, antibody cross-reactivity andtumour cell killing activity. The expression product of thesynthetic gene has been purified to homogeneity by a two-stepion-exchange procedure and the purified material shown to beactive.     

Effect of replacing helical glycine residues with alanines on reversible and irreversible stability and production of Aspergillus awamori glucoamylase

To decrease irreversible thermoinactivation of Aspergillus awamoriglucoamylase, five Gly residues causing helix flexibility werereplaced with Ala residues. Mutation of Gly57 did not affectthermostability. Mutation of Gly137 doubled it at pHs 3.5 and4.5 but barely changed it at pH 5.5. The Gly139<IMG SRC="http://peds.oxfordjournals.org/math/rarr.gif" ALT="->" BORDER="0">Ala mutationdid not change thermostability at pH 3.5, improved it at pH4.5 and worsened it at pH 5.5. The Gly137/Gly139<IMG SRC="http://peds.oxfordjournals.org/math/rarr.gif" ALT="->" BORDER="0">Ala/Ala mutationgave 1.5–2-fold increased thermostabilities at pHs 3.5–5.5.Mutations of Gly251 and Gly383 decreased it at all pHs. Gly137<IMG SRC="http://peds.oxfordjournals.org/math/rarr.gif" ALT="->" BORDER="0">Alaand Gly137/Gly139<IMG SRC="http://peds.oxfordjournals.org/math/rarr.gif" ALT="->" BORDER="0">Ala/Ala glucoamylases are the most stable yetproduced by mutation. Guanidine treatment at pH 4.5 decreasedthe reversible stabilities of Gly137<IMG SRC="http://peds.oxfordjournals.org/math/rarr.gif" ALT="->" BORDER="0">Ala, Gly139<IMG SRC="http://peds.oxfordjournals.org/math/rarr.gif" ALT="->" BORDER="0">Ala and Gly137/Gly139<IMG SRC="http://peds.oxfordjournals.org/math/rarr.gif" ALT="->" BORDER="0">Ala/Alaglucoamylases at infinite dilution while not changing thoseof Gly251<IMG SRC="http://peds.oxfordjournals.org/math/rarr.gif" ALT="->" BORDER="0">Ala and Gly 383<IMG SRC="http://peds.oxfordjournals.org/math/rarr.gif" ALT="->" BORDER="0">Ala glucoamylases, which is, in general,opposite to what occurred with thermoinactivation. Mutationof Gly57 greatly improved the extracellular glucoamylase productionby yeast, that of Gly137 barely affected it and those of Gly139and of both Gly137 and Gly139 strongly impeded it. These observationssuggest that <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helix rigidity can affect reversible and irreversibleglucoamylase stability differently, that the effects of multiplemutations within one <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helix to improve stability are not alwaysadditive and that even single mutations can strongly affectextracellular enzyme production.