Isa Gokce

Voltage gating is a fundamental feature of porin and toxin β‐barrel membrane channels

Beta‐barrel pores are found in outer membrane porins of Gram‐negative bacteria, bacterial toxins and mitochondrial channels. Apart from the β‐barrel the three groups show no close sequence or structural homology but these pores exhibit symmetrical voltage gating when reconstituted into planar lipid bilayers. The structures of several of these are known and many site‐directed mutants have been examined. As a result it seems evident that the gating is a common characteristic of these unrelated large pores and is not generated by specialised structures in the pore lumen.

Expression of proteins using the third domain of the Escherichia coli periplasmic-protein TolA as a fusion partner

The third domain of the periplasmic protein TolA from Escherichia coli (TolAIII) was used as a fusion partner in the expression of various proteins from bacteria and eukaryotes. TolAIII is small domain, expressed in high yields as a soluble protein in the cytoplasm of E. coli. Proteins were linked to the C-terminus of TolAIII by a short flexible linker containing sites for endopeptidases. Three different vectors were prepared, containing sites for enterokinase, thrombin or factor Xa. Fusion proteins also contain a His(6)-Ser(2) tag at their N-terminus for easier purification. Up to 90 mg fusion protein per liter bacterial culture was obtained using these vectors. Colicin N R-domain was expressed with this system as a fusion and processed further for functional studies. The yield of final pure R-domain was doubled as compared to the direct expression. The system may prove to be useful in the preparation of other peptides and proteins.

Determination of Antioxidant Activity of Marshmallow Flower (Althaea officinalis L.)

Abstract The antioxidant properties of marshmallow (Althaea officinalis L., Fam. Malvaceae) ethanolic extract was evaluated using different antioxidant tests, including reducing power, free radical scavenging, superoxide anion radical scavenging, and metal chelating activities. The extract of marshmallow (A. officinalis L.) exhibited strong total antioxidant activity. The concentration of 50, 100, and 250 µg/mL of ethanol extract of marshmallow (A. officinalis L.) showed 85.5%, 91.2%, and 96.4% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, 100 µg/mL of standard antioxidant such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and α‐tocopherol exhibited 94.5%, 99.1%, and 80% inhibition on peroxidation of linoleic acid emulsion, respectively. The extract of marshmallow (A. officinalis L.) had effective reducing power, free radical scavenging, superoxide anion radical scavenging, and metal chelating activities at same concentration (50, 100, and 250 µg/mL). Those various antioxidant activities were compared with standard antioxidants such as BHA, BHT, and α‐tocopherol.

A Natively Unfolded Toxin Domain Uses Its Receptor as a Folding Template

Natively unfolded proteins range from molten globules to disordered coils. They are abundant in eukaryotic genomes and commonly involved in molecular interactions. The essential N-terminal translocation domains of colicin toxins from Escherichia coli are disordered bacterial proteins that bind at least one protein of the Tol or Ton family. The colicin N translocation domain (ColN-(1–90)), which binds to the C-terminal domain of TolA (TolA-(296–421)), shows a disordered far-UV CD spectrum, no near-UV CD signal, and non-cooperative thermal unfolding. As expected, TolA-(296–421) displays both secondary structure in far-UV CD and tertiary structure in near-UV CD. Furthermore it shows a cooperative unfolding transition at 65 °C. CD spectra of the 1:1 complex show both increased secondary structure and colicin N-specific near-UV CD signals. A new cooperative thermal transition at 35 °C is followed by the unchanged unfolding behavior of TolA-(296–421). Fluorescence and surface plasmon resonance confirm that the new unfolding transition accompanies dissociation of ColN-(1–90). Hence upon binding the disordered structure of ColN-(1–90) converts to a cooperatively folded domain without altering the TolA-(296–421) structure.

High level expression of His-tagged colicin pore-forming domains and reflections on the sites for pore formation in the inner membrane

There exists ample evidence for the assumption that pore-forming colicins cannot exert their toxicity within the producing cell and that they must gain access to the outer face of the cytoplasmic membrane to achieve this. We wished to construct pET-vectors to produce pore-forming domains of colicin A and N with N-terminal hexa-histidine tags under the control of a T7 promoter. This was only possible when the correct immunity protein was also present. Hence it appears that this system exhibits the peculiarity that there is a toxicity associated with the over produced pore-forming domain. However, when the ratio of colicin to immunity protein is compared it is still clear that direct insertion into the cytoplasmic membrane does not occur and that membrane translocation of the colicin at limited sites may be occurring. This article reviews previous literature on the subject in terms of a model for limited sites of colicin action.

High-yield expression and purification of soluble forms of the anti-apoptotic Bcl-xL and Bcl-2 as TolAIII-fusion proteins

A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.

Stabilising and destabilising modifications of cysteines in the E. coli outer membrane porin protein OmpC

Three sulfhydryl labels were used to modify two mutated sites, R37C and R74C in the eyelet of the outer membrane porin OmpC. Modification of R37C with the neutral groups Aldrithiol and bimane increases thermal stability but the negatively charged iodoacetate causes a decrease in thermal stability. The effects of substitution at R74C were less significant. Bimane labelling increases the voltage sensitivity and decreases the single channel conductance at R37C asymmetrically with smaller channels being recorded at cis negative voltages. Negatively charged acetate does not affect the voltage gating.

Expression analysis of metallothioneins and mineral contents in tomato (Lycopersicon esculentum) under heavy metal stress.

BACKGROUND Heavy metals are considered to be the most important pollutants in the contamination of soils; they adversely affect plant growth and development and cause some physiological and molecular changes. The contamination of agricultural soils by heavy metals has changed the mineral element content of vegetables. Plant metallothioneins (MTs) are thought to have the functional role in heavy metal homeostasis, and they are used as the biomarkers for evaluating environmental pollution. We aimed to evaluate the expression of MT isoforms (MT1, 2, 3 and 4) and some mineral element composition of tomato roots, leaves and fruits exposed to copper and lead. RESULTS Heavy metal applications increased MT1 and MT2 gene expressions compared to the control in the tissues of tomato. The highest level of MT1 and MT2 transcripts was found in roots and leaves, respectively. The expression of MT3 is induced in roots, leaves and fruits except for Pb treatment in roots. MT4 expression increased in fruits; however, other tissues did not show a clear change. Our results indicated that Cu content was higher than Pb in all tissues of tomato. The lower doses of Cu (10 ppm) increased the content of Mg, Fe, Ca and Mn in roots. Pb generally increased the level of minerals in leaves and fruits, but it decreased Mg, Mn and Fe contents in roots. CONCLUSION Both heavy metals not only moved to aerial parts but also caused alterations to mineral element levels. These results show that MT transcripts are regulated by Cu and Pb, and expression pattern changes to MT isoforms and tissue types.

Bacillus subtilis’e Ait Ksilanaz (xyn-akky1)’in Escherichia coli’de Klonlanması, Ekspresyonu ve Karakterizasyonu

Bu calismada, Bacillus subtilis akky1 straini Turkiye’nin Ordu Ili’nin Akkus ilcesindeki kayin ormanindan alinan topraktan izole edilmistir. akky1 straini 16S rRNA analizi ile tanimlanmistir. 16S rRNA dizisi Bacillus subtilis strain B7 (KC310823.1) ile %100’luk bir benzerlik gostermistir. GenBank’da verilen Bacillus subtilis ksilanazina ait gen dizisi esas alinarak tasarlanan primerler kullanilarak genomic DNA’dan 642 bp’lik bir DNA fragmenti elde edilmistir. Ksilanazi kodlayan gen, pET28b (+) ekspresyon vektorune klonlanmis ve Escherichia coli BL21 (DE3)’de ekpresse edilmistir. Rekombinant olarak uretilen protein nikel afinite kromatografisi ile saflastirilmis ve ksilanaz aktivitesi belirlenmistir. Saflastirilan ksilanazin molekuler agirligi SDS-PAGE ile 26.0 kDa olarak belirlenmistir. Rekombinant enzimin optimum pH’si 6.0 ve sicakligi 60°C, Km degeri ise 3.33 mg/ml olarak tespit edilmistir.

Expression, purification, and characterization of bovine chymosin enzyme using an inducible pTOLT system

ABSTRACT In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The different areas where enzymes are used and their economic value of biotechnological products further increases their importance. There are hundreds of different types of cheese but each is made by coagulating milk using rennet to give curds. Today, researchers have begun to develop alternative systems in the cheese industry related to milk-clotting enzymes. In this study, the nucleic acid sequence encoding the optimized chymosin enzyme was used and cloned by Not I and Mlu I restriction enzymes into pTOLT vector system. Then using this construct, the enzyme as a fusion with Tol-A-III protein was produced in Escherichia coli BL21 (DE3) cells. After disrupting the E. coli cell and separating from the constituents by high speed centrifugation, the enzyme was purified by affinity chromatography and fractions were analyzed by SDS–PAGE. Purified enzyme has shown its activity. Optimum temperature and pH of CHY-Tol-A-III protein were 40°C and 6.5, respectively.