Biochemical characterization of recombinant 20s proteasome from thermoplasma volcanium and cloning of it's regulatory subunit gene

Baydar, Gözde
In this study, we have characterized some biochemical and electrophoretic features of recombinant 20S Proteasome from a thermoacidophilic archaeon Thermoplasma volcanium. As revealed by SDS-PAGE the 20S Proteasome was composed of two subunits, ?- and β- subunits with estimated molecular masses of 24 kDa and 23 kDa, respectively. The highest chymotryptic activity was observed over an alkaline pH range (pH 8.0 ا pH 9.0) and the optimum temperature for the activity was determined as 85oC. The heat stability of proteasome was quite high after treatment at 98oC for 30 minutes, 64 % of the activity has still been retained. The highest activity associated with the Thermoplasma volcanium proteasome was found to be peptidylglutamyl peptidase activity. Within the scope of this project, also, we have cloned a 26S Proteasome related Regulatory Subunit gene of Thermoplasma volcanium. For cloning we have followed a PCR based approach. Amplification of 26S Proteasome Regulatory Subunit gene from chromosomal DNA of Tp. volcanium yielded a product of 1419 bp containing an open reading frame of 1128 bp comprising the structural gene. The PCR amplicon was cloned using pDrive vector in E.coli TG-1 cells. Out of ten putative recombinants, three plasmids, E.coli pD1-6, E.coli pD2-3, E.coli pD3-1, were proved to be true recombinants and selected for further characterization by restriction mapping and expression studies. ATPase activities of cell free extracts from both recombinant and non-recombinant E.coli strains were measured and found that ATPase activities in cell free extracts of recombinant strains were 10 times higher than non-recombinants. This result indicates sucessful expression of the cloned regulatory subunit gene with ATPase activity in E.coli.


PCR cloning and heterologous expression of Scytalidium thermophilum laccase gene in Aspergillus sojae
Koçlar, Gülden; Ögel, Zümrüt Begüm; Department of Biotechnology (2005)
In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding ...
Molecular recognition of poly(A) by small ligands: an alternative method of analysis reveals nanomolar, cooperative and shape-selective binding
Persil Çetinkol, Özgül (Oxford University Press (OUP), 2009-02-01)
A few drug-like molecules have recently been found to bind poly(A) and induce a stable secondary structure (T(m)approximate to 60 degrees C), even though this RNA homopolymer is single-stranded in the absence of a ligand. Here, we report results from experiments specifically designed to explore the association of small molecules with poly(A). We demonstrate that coralyne, the first small molecule discovered to bind poly(dA), binds with unexpectedly high affinity (K(a) >10(7) M(-1)), and that the crescent sh...
Molecular dynamics simulations and coupled nucleotide substitution experiments indicate the nature of A center dot A base pairing and a putative structure of the coralyne-induced homo-adenine duplex
Joung, In Suk; Persil Çetinkol, Özgül; HUD, Nicholas V.; Cheatham, Thomas E. (Oxford University Press (OUP), 2009-12-01)
Coralyne is an alkaloid drug that binds homo-adenine DNA (and RNA) oligonucleotides more tightly than it does Watson-Crick DNA. Hud's laboratory has shown that poly(dA) in the presence of coralyne forms an anti-parallel duplex, however attempts to determine the structure by NMR spectroscopy and X-ray crystallography have been unsuccessful. Assuming adenine-adenine hydrogen bonding between the two poly(dA) strands, we constructed 40 hypothetical homo-(dA) anti-parallel duplexes and docked coralyne into the s...
Functional characterization of microrna-125b expression in MCF7 breast cancer cell line
Tuna, Serkan; Erson Bensan, Ayşe Elif; Department of Biology (2010)
microRNA dependent gene expression regulation has roles in diverse processes such as differentiation, proliferation and apoptosis. Therefore, deregulated miRNA expression has functional importance for various diseases, including cancer. miR-125b is among the commonly downregulated miRNAs in breast cancer cells . Therefore we aimed to characterize the effects of miR-125b expression in MCF7 breast cancer cell line (BCCL) to better understand its roles in tumorigenesis. Here, we investigated mir-125 family mem...
Cloning and characterization of industrially important alpha-galactosidase genes from the human pathogen aspergillus fumigatus
Söyler, U. Betül; Ögel, Zümrüt Begüm; Department of Food Engineering (2004)
In this study, molecular cloning studies were performed on the a-galactosidase genes of Aspergillus fumigatus IMI 385708. This organism is an opportunistic saprophytic fungus and a human pathogen, mainly affecting immunocompromised patients. A. fumigatus is a thermotolerant fungus and can efficiently produce thermostable a-galactosidase. Two different cloning strategies were undertaken in this study. A. fumigatus cDNA library, prepared previously, was screened with three different probes. No net results wer...
Citation Formats
G. Baydar, “Biochemical characterization of recombinant 20s proteasome from thermoplasma volcanium and cloning of it’s regulatory subunit gene,” M.S. - Master of Science, Middle East Technical University, 2006.