scholarly journals Cloning, expression and purification of functionally active Saccharomyces cerevisiae Polo-like Kinase, Cdc5 in E. coli

2016 ◽  
Vol 23 (3) ◽  
pp. 410-419 ◽  
Author(s):  
Ajamaluddin Malik ◽  
Abdulrahman M. Alsenaidy ◽  
Mohamed Elrobh ◽  
Wajahatullah Khan ◽  
Mohammed S. Alanazi ◽  
...  

2013 ◽  
Vol 288 (20) ◽  
pp. 13951-13959 ◽  
Author(s):  
Yan Zhang ◽  
Xiuxiang An ◽  
JoAnne Stubbe ◽  
Mingxia Huang

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.


Author(s):  
Alequis Tomás Pavón Oro

El cáncer es la segunda causa de muerte en el mundo, y específicamente en Chile el cáncer colorrectal es el único que presenta un aumento sostenido de la mortalidad en la última década. La búsqueda de nuevos agentes quimioterapeúticos anticancerígenos ha propuesto a los microorganismos extremófilos como una fuente potencial para obtener moléculas citotóxicas, que induzcan apoptosis en las células tumorales. Las condiciones extremas del continente antártico y las presiones selectivas por el espacio y los nutrientes que se producen entre los microorganismos del rizobioma de la planta Deschampsia antarctica Desv sugirieron como hipótesis que las bacterias rizosféricas aisladas en la Antártica secretan al sobrenadante de cultivo moléculas bioactivas que inhiben la invasión y proliferación de líneas tumorales humanas de origen colorrectal mediante un mecanismo apoptótico. En este sentido, el objetivo general del trabajo fue identificar y caracterizar a moléculas bioactivas con acción antinvasiva y antiproliferativa, además, determinar el mecanismo inhibitorio de la proliferación en líneas tumorales humanas de origen colorrectal. Los resultados del primer objetivo específico demostraron que los sobrenadantes de cultivo de los aislados rizosféricos antárticos K2 y MI disminuyeron la viabilidad de la línea celular de adenocarcinoma colorrectal LoVo en el ensayo de reducción metabólica del MTT. Además, como los sobrenadantes no tuvieron efecto en la viabilidad de las bacterias E. coli y Staphylococcus aureus, y tampoco en los hongos unicelulares Candida albicans y Saccharomyces cerevisiae, el resultado indicó que la actividad antiproliferativa fue selectiva hacia la línea celular LoVo.


2014 ◽  
Vol 28 (S1) ◽  
Author(s):  
Angel Diaz Martinez ◽  
Jose Renato Cussiol ◽  
Marcus Smolka

OALib ◽  
2015 ◽  
Vol 02 (03) ◽  
pp. 1-5
Author(s):  
Nora Guida ◽  
Marcela Mascolo ◽  
Mariano Laino ◽  
Carla Bustos ◽  
Pablo Franco

2020 ◽  
Vol 11 (3) ◽  
pp. 4353-4358
Author(s):  
Saripah Salbiah Syed Abdul Azziz ◽  
Ahmed Kareem Obaid Aldulaimi ◽  
Saadon Abdulla Aowda ◽  
Yuhanis Mhd Bakri ◽  
Ali Arkan Majhool ◽  
...  

The study aimed to isolate and identify the phytochemical components of Polyalthia lateriflora leaves and evaluate the antimicrobial activity. Six well-known compounds, including three triterpene lupeol (1) betulinic acid (2), β-Sitosterol-β-D-glucoside (3) and three oxoaporphine alkaloids O-methylmoschotaline (4), liriodenine (5) and atherosperminine (6). Structural elucidation of compounds were established through spectroscopic techniques such as 1D and 2D NMR (1H and 13C, DEPT, COSY, NOESY, HMBC, HMQC), IR and LC-MS. The isolated compounds and crude extracted were tested for their antibacterial potential against several microorganisms including P. aeruginosa, E. coli, s, S. aureus, B. subtilis and Saccharomyces cerevisiae and its showed significant inhibition toward the organisms species with different concentration range.


2015 ◽  
Vol 16 (1) ◽  
pp. 31
Author(s):  
Kusdianawati Kusdianawati ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Bugi Ratno Budiarto ◽  
Fatimah Fatimah ◽  
...  

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from <em>Lactobacillus plantarum</em> S34 in <em>Escherichia coli</em>. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. <em>plantarum</em> S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. <em>plantarum</em> S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. <em>coli</em> BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.


Gene Reports ◽  
2019 ◽  
Vol 16 ◽  
pp. 100417 ◽  
Author(s):  
Abbas Shapouri Moghaddam ◽  
Kiarash Ghazvini ◽  
Abbas Bahador ◽  
Mohammad Derakhshan ◽  
Azad Khaledi

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