Lantibiotic Nisin and Its Detection Methods

The type A lantibiotic nisin produced by several Lactococcus lactis strains, and one Streptococcus uberis strainis a small antimicrobial peptide that inhibits the growth of a wide range of gram-positive bacteria, such as Bacillus, Clostridium, Listeria and Staphylococcus species. It is nontoxic to humans and used as a food preservative (E234) in more than 50 countries including the EU, the USA, and China. National legislations concerning maximum addition levels of nisin in different foods vary greatly. Therefore, there is a demand for non-laborious and sensitive methods to identify and quantify nisin reliably from different food matrices. The horizontal inhibition assay, based on the inhibitory effect of nisin to Micrococcus luteus is the base for most quantification methods developed so far. However, the sensitivity and accuracy of the agar diffusion method is affected by several parameters. Immunological tests have also been described. Taken into account the sensitivity of immunological methods to interfering substances within sample matrices, and possible cross-reactivities with lantibiotics structurally close to nisin, their usefulness for nisin detection from food samples remains limited. The proteins responsible for nisin biosynthesis, and producer self-immunity are encoded by genes arranged into two inducible operons, nisA/Z/QBTCIPRK and nisFEG, which also contain internal, constitutive promoters PnisI and PnisR. The transmembrane histidine kinase NisK and the response regulator NisR form a two-component signal transduction system, in which NisK autophosphorylates after exposure to extra cellular nisin, and subsequently transfers the phosphate to NisR. The phosphorylated NisR then relays the signal downstream by binding to two regulated promoters in the nisin gene cluster, i.e the nisA/Z/Qand the nisF promoters, thus activating transcription of the structural gene nisA/Z/Q and the downstream genes nisBTCIPRK from the nisA/Z/Q promoter, and the genes nisFEG from the nisF promoter. In this work two novel and highly sensitive nisin bioassays were developed. Both of these quantification methods were based on NisRK mediated, nisin induced Green Fluorescent Protein (GFP) fluorescence. The suitabilities of these assays for quantifica¬tion of nisin from food samples were evaluated in several food matrices. These bioassays had nisin sensitivities in the nanogram or picogram levels. In addition, shelf life of nisin in cooked sausages and retainment of the induction activity of nisin in intestinal chyme (intestinal content) was assessed.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

L-rhamnose-1-dehydrogenase gene and L-rhamnose catabolism in the yeast Pichia stipitis

The purpose of this work was to identify some of the genes of the catabolic route of L-rhamnose in the yeast Pichia stipitis. There are at least two distinctly different pathways for L-rhamnose catabolism. The one described in bacteria has phosphorylated intermediates and the enzymes and the genes of this route have been described. The pathway described in yeast does not have phosphorylated intermediates. The intermediates and the enzymes of this pathway are known but none of the genes have been identified. The work was started by purifying the L-rhamnose dehydrogenase, which oxidates L-rhamnose to rhamnonic acid-gamma-lactone. NAD is used as a cofactor in this reaction. A DEAE ion exchange column was used for purification. The active fraction was further purified using a non-denaturing PAGE and the active protein identified by zymogram staining. In the last step the protein was separated in a SDS-PAGE, the protein band trypsinated and analysed by MALDI-TOF MS. This resulted in the identification of the corresponding gene, RHA1, which was then, after a codon change, expressed in Saccharomyces cerevisiae. Also C- or N-terminal histidine tags were added but as the activity of the enzyme was lost or strongly reduced these were not used. The kinetic properties of the protein were analysed in the cell extract. Substrate specifity was tested with different sugars; L-rhamnose, L-lyxose and L-mannose were oxidated by the enzyme. Vmax values were 180 nkat/mg, 160 nkat/mg and 72 nkat/mg, respectively. The highest affinity was towards L-rhamnose, the Km value being 0.9 mM. Lower affinities were obtained with L-lyxose, Km 4.3 mM, and L-mannose Km 25 mM. Northern analysis was done to study the transcription of RHA1 with different carbon sources. Transcription was observed only on L-rhamnose suggesting that RHA1 expression is L-rhamnose induced. A RHA1 deletion cassette for P. stipitis was constructed but the cassette had integrated randomly and not targeted to delete the RHA1 gene. Enzyme assays for L-lactaldehyde dehydrogenase were done similarly to L-rhamnose dehydrogenase assays. NAD is used as a cofactor also in this reaction where L-lactaldehyde is oxidised to L-lactate. The observed enzyme activities were very low and the activity was lost during the purification procedures.

Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6•0–6•5, when the pH of the external medium was varied between 2•3–7•0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn2+ ions partially protected the enzyme against this inactivation. Mn2+-dependent glutamine synthetase activity was higher at acidic pH (6•0) compared to Mg2+-supported activity. While the concentration of Mg2+ required to optimally activate glutamine synthetase at pH 6•0 was very high (≥ 50 mM), Mn2+ was effective at 4 mM. Higher concentrations of Mn2+ were inhibitory. The inhibition of both Mn2+ and Mg2+-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn2+ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis.

Genetic conservation of Phosphine Resistance in the Rice Weevil Sitophilus oryzae (L.)

High levels of resistance to phosphine in the rice weevil Sitophilus oryzae have been detected in Asian countries including China and Vietnam, however there is limited knowledge of the genetic mechanism of resistance in these strains. We find that the genetic basis of strong phosphine resistance is conserved between strains of S. oryzae from China, Vietnam and Australia. Each of four strongly resistant strains has an identical amino acid variant in the encoded dihydrolipoamide dehydrogenase (DLD) enzyme that was previously identified as a resistance factor in Rhyzopertha dominica and Tribolium castaneum. The unique amino acid substitution, Asparagine > Threonine (N505T) of all strongly resistant S. oryzae corresponds to the position of an Asparagine > Histidine variant (N506H) that was previously reported in strongly resistant R. dominica. Progeny (F16 and F18) from two independent crosses showed absolute linkage of N505T to the strong resistance phenotype, indicating that if N505T was not itself the resistance variant that it resided within 1 or 2 genes of the resistance factor. Non-complementation between the strains confirmed the shared genetic basis of strong resistance, which was supported by the very similar level of resistance between the strains, with LC50 values ranging from 0.20 to 0.36 mgL-1 for a 48 hour exposure at 25°C. Thus, the mechanism of high level resistance to phosphine is strongly conserved between R. dominica, T. castaneum and S. oryzae. A fitness cost associated with strongly resistant allele was observed in segregating populations in the absence of selection.

Chemical modification studies on Abrus agglutinin Involvement of tryptophan residues in sugar binding

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydratebinding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2- hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.

The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Mammalian spermatid specific protein, TP2, is a zinc metalloprotein with two finger motifs

An analysis of the recently reported cDNA derived amino acid sequences of mouse (Kleene and Flynn, J. Biol. Chem. , 17272–17277, 1987) and rat (Luersson Image ,Nucl. Acids Res. Image , 3585, 1989). TP2 has revealed the presence of two potential zinc finger motifs involving cysteine and histidine residues. TP2, as purified from rat elongating spermatids, is shown here to contain 0.2 atoms of zinc bound per molecule of the protein by atomic absorption spectroscopy. On incubation with 10 μM ZnCl2, Image , and subsequent exhaustive dialysis, TP2 had 2 atoms of zinc bound per molecule. The involvement of cysteine residues of TP2 in coordination with zinc was also suggested by the observation that TP2 could be labeled, Image , with iodoacetamidofluorescein only after preincubation of spermatid nuclei with EDTA. The zinc finger domains of TP2 may play an important role in initiation of chromatin condensation and /or cessation of transcriptional activity during mammalian spermiogenesis. DTT, Dithiothreitol; IAF, Iodoacetamido-fluorescein; SDS, Sodium dodecyl sulfate; PAGE, Polyacrylamide gel electrophoresis; PMSF, Phynyl methyl sulfonyl fluoride

Cytokinin signalling in the regulation of cambial development

Secondary growth of plants is of pivotal importance in terrestrial ecosystems, providing a significant carbon sink in the form of wood. As plant biomass accumulation results largely from the cambial growth, it is surprising that quite little is known about the hormonal or genetic control of this important process in any plant species. The central aim of my thesis studies was to explore the function of cytokinin in the regulation of cambial development. Since their discovery as regulators of plant cell divisions, cytokinins have been assumed to participate in the control of cambial development. Evidence for this action was deduced from hormone treatment experiments, where exogenously applied cytokinin was shown to enhance cambial cell divisions in diverse plant organs and species. In my thesis work, the conservation of cytokinin signalling and homeostasis genes between a herbaceous plant, Arabidopsis, and a hardwood tree species, Populus trichocarpa. Presumably reflecting the ancient origin of cytokinin signalling system, the Populus genome contains orthologs for all Arabidopsis cytokinin signalling and homeostasis genes. Thus, genes belonging to five main families of isopentenyl transferases (IPTs), cytokinin oxidases (CKXs), two-component receptors, histidine containing phosphotransmitters (HPts) and response regulators (RRs) were identified from the Populus genome. Three subfamilies associated with cytokinin signal transduction, the CKI1-like family of two-component receptors, the AHP4-like HPts, and the ARR22-like atypical RRs, were significantly larger in Populus genome than in Arabidopsis. Potential contribution to the extensive secondary development of Populus by the members of these considerably expanded gene families will be discussed. Representatives of all cytokinin signal transduction elements were expressed in the Populus cambial zone, and most of the expressed genes appeared to be slightly more abundant on the phloem side of the meristem. The abundance of cytokinin related genes in the cambium emphasizes the important role of this hormone in the regulation of the extensive secondary growth characteristic of tree species. The function of the pseudo HPts in primary vascular development was studied in Arabidopsis root vasculature. It was demonstrated that the pseudo HPt AHP6 has a role in locally inhibiting cytokinin signalling in the protoxylem position in the Arabidopsis root, thus enabling differentiation of the protoxylem cell file. The possible role of pseudo HPts in cambial development will be discussed. The expression peak of cytokinin signalling genes in the tree cambial zone strongly indicates that cytokinin has a role in the regulation of this meristem function. To address whether cytokinin signalling is required for cambial activity, transgenic Populus trees with modified cytokinin signalling were produced. These trees were expressing a cytokinin catabolic gene from Arabidopsis, CYTOKININ OXIDASE 2, (AtCKX2) under the promoter of a Betula CYTOKININ RECEPTOR 1 (BpCRE1). The pBpCRE1::CKX2 transgenic Populus trees showed a reduced concentration of a biologically active cytokinin, correlating with their impaired cytokinin response. Furthermore, the radial growth of these trees was compromised, as illustrated by a smaller stem diameter than in wild-type trees of the same height. Moreover, the level of cambial cytokinin signalling was down-regulated in these thin-stemmed trees. The reduced signalling correlated with a decreased number of meristematic cambial cells, implicating cytokinin activity as a direct regulator of cambial cell division activity. Together, the results of my study indicate that cytokinins are major hormonal regulators required for cambial development.

Sequence Specific Interaction between Promoter DNA and Escherichia coli RNA Polymerase: Comparative Thermodynamic Analysis with One Immobilized Partner

Sequence specific interaction between DNA and protein molecules has been a subject of active investigation for decades now. Here, we have chosen single promoter containing bacteriophage Delta D-III T7 DNA and Escherichia coli RNA polymerase and followed their recognition at the air-water interface by using the surface plasmon resonance (SPR) technique, where the movement of one of the reacting species is restricted by way of arraying them on an immobilized support. For the Langmuir monolayer studies, we used a RNA polymerase with a histidine tag attached to one of its subunits, thus making it an xcellent substrate for Ni(II) ions, while the SPR Studies were done using biotin-labeled DNA immobilized on a streptavidin-coated chip. Detailed analysis of the thermodynamic parameters as a function of concentration and temperature revealed that the interaction of RNA polymerase with T7 DNA is largely entropy driven (83 (+/- 12) kcal mol(-1)) with a positive enthalpy of 13.6 (+/- 3.6) kcal mol(-1), The free energy of reaction determined by SPR and Langmuir-Blodgett technique was -11 (+/- 2) and -15.6 kcal mol(-1), respectively. The ability of these methods to retain the specificity of the recognition process was also established.

Characterization of the Entamoeba histolytica Ornithine Decarboxylase-Like Enzyme

Background: The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme. Methodology and Results: To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC)-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF) of similar to 1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa) from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size similar to 46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids-aspartate-332, aspartate-361, and tyrosine-323-by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity. Conclusion: To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from E. histolytica. Computer modeling revealed that three of the critical residues required for binding of DFMO to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO.

A network representation of protein structures: Implications for protein stability

This study views each protein structure as a network of noncovalent connections between amino acid side chains. Each amino acid in a protein structure is a node, and the strength of the noncovalent interactions between two amino acids is evaluated for edge determination. The protein structure graphs (PSGs) for 232 proteins have been constructed as a function of the cutoff of the amino acid interaction strength at a few carefully chosen values. Analysis of such PSGs constructed on the basis of edge weights has shown the following: 1), The PSGs exhibit a complex topological network behavior, which is dependent on the interaction cutoff chosen for PSG construction. 2), A transition is observed at a critical interaction cutoff, in all the proteins, as monitored by the size of the largest cluster (giant component) in the graph. Amazingly, this transition occurs within a narrow range of interaction cutoff for all the proteins, irrespective of the size or the fold topology. And 3), the amino acid preferences to be highly connected (hub frequency) have been evaluated as a function of the interaction cutoff. We observe that the aromatic residues along with arginine, histidine, and methionine act as strong hubs at high interaction cutoffs, whereas the hydrophobic leucine and isoleucine residues get added to these hubs at low interaction cutoffs, forming weak hubs. The hubs identified are found to play a role in bringing together different secondary structural elements in the tertiary structure of the proteins. They are also found to contribute to the additional stability of the thermophilic proteins when compared to their mesophilic counterparts and hence could be crucial for the folding and stability of the unique three-dimensional structure of proteins. Based on these results, we also predict a few residues in the thermophilic and mesophilic proteins that can be mutated to alter their thermal stability.

Crystal structure of dimeric FabZ of Plasmodium falciparum reveals conformational switching to active hexamers by peptide flips

The crystal structure of beta-hydroxyacyl acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) has been determined at a resolution of 2.4 angstrom. PfFabZ has been found to exist as a homodimer (d-PfFabZ) in the crystals of the present study in contrast to the reported hexameric form (h-PfFabZ) which is a trimer of dimers crystallized in a different condition. The catalytic sites of this enzyme are located in deep narrow tunnel-shaped pockets formed at the dimer interface. A histidine residue from one subunit of the dimer and a glutamate residue from the other subunit lining the tunnel form the catalytic dyad in the reported crystal structures. While the position of glutamate remains unaltered in the crystal structure of d-PffabZ compared to that in b-PfFabZ, the histidine residue takes up an entirely different conformation and moves away from the tunnel leading to a His-Phe cis-trans peptide flip at the histidine residue. In addition, a loop in the vicinity has been observed to undergo a similar flip at a Tyr-Pro peptide bond. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate binding. The dimeric state and an altered catalytic site architecture make d-PfFabZ distinctly different from the FabZ structures described so far. Dynamic light scattering and size exclusion chromatographic studies clearly indicate a pH-related switching of the dimers to active hexamers. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserv.

The regulation of nitrate reductas enad catalase by amino acids in Neurospora crassa

The induction of nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) by nitrate in Neurospora crassa and its control by amino acids have been studied. The growth-inhibitory amino acids, isoleucine and cysteine as well as the growth-promotory ones, glutamine, asparagine, arginine, histidine and NH4+, repress nitrate reductase effectively. Methionine, tryptophan, proline, aspartic acid and glutamic acid exert little control on nitrate reductase. The repression of nitrate reductase by cysteine, isoleucine, glutamine and asparagine is accompanied by inactivation of the enzyme present initially. The nitrate-induced NADPH-cytochrome c reductase (NADPH:cytochrome c oxidoreductase, EC 1.6.2.3) is also repressed by amino acids which control nitrate reductase, providing further evidence to show that these two enzyme activities may reside in the same protein. Catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) has been found to be induced subsequent to the induction of nitrate reductase by nitrate in N. crassa. The induction of catalase is probably by its substrate H2O2 which would be formed by the interaction of the flavine component of nitrate reductase with oxygen. The amino acids which control nitrate reductase, repress catalase also. The catalase level appears to be determined by the nitrate reductase activity of the mycelia.

Cloning,overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from scherichia coli

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively. (C) 2010 Elsevier Inc. All rights reserved.