Expression of recombinant acid protease (thermopsin) gene from Thermoplasma volcanium

Download

index.pdf

Date

2006

Author

Koyuncu, Bilsev

Metadata

Show full item record

Item Usage Stats

246
views

73
downloads

Acid proteases, commonly known as aspartic proteases are degredative enzymes which catalyze the cleavage reaction of peptide bonds in proteins with a pH optimum in the acidic range (pH 3-4). Acid proteases have crucial roles in metabolism. Moreover, they are used in different fields of industry. Thermophilic microorganisms, especially archaea, gain special interest because of their thermal stability for both fundamental and industrial researches. Thermopsin is an extracellular acid protease and a member of A5 family of proteases. This thermophilic enzyme has no characteristic active aspartyl residue, is insensitive to pepstatin and no apparent sequence homology to other acid proteases and therefore represents a new class of acid proteases. Thermophilic archaeal strain Thermoplasma volcanium GSS1 (optimum temperature 550C and pH 2.7) in the genome has a putative thermopsin gene encoding 998 amino acid enzyme. In this study thermopsin gene from Thermoplasma volcanium was expressed in E. coli as fusion with 6xHis tag under the control of T5 transcription/translation system. Putative thermopsin gene from Thermoplasma volcanium was amplified by PCR method using two primer sets and cloned. A 3080 bp and a 3070bp PCR products were obtained by using TP1/TP2 primer set (thermopsin gene with the start codon) and TP1̕/TP2 primer set (thermopsin gene missing start codon) respectively. PCR amplified thermopsin genes pDrive and pUC18 vectors in E. coli TG1 were cloned using and then cloned genes were sub-cloned directionally into pQE triple vector set for expression. In these expression vectors, cloned genes are placed downstream of a 6XHis tag to produce an expression fusion. E.coli strains (M15[pREP4], SG13009[pREP4], and TG1) used as hosts. Recombinant colonies screened by colony blot/hybridization method based on immunological detection of the expressed 6XHis tag fusion by

Subject Keywords

Genetics.

URI

http://etd.lib.metu.edu.tr/upload/3/12606995/index.pdf
https://hdl.handle.net/11511/15841

Collections

Graduate School of Natural and Applied Sciences, Thesis

Suggestions

OpenMETU
Core

Cloning and expression of periplasmic (clp p-like) and membrane-bound serine protease genes of thermoplasma volcanium in escherichia coli Demirok, Burçak; Kocabıyık, Semra; Department of Biology (2006) Serine proteases are a family of proteases that utilize an activated serine residue in the substrate-binding pocket to catalytically hydrolyze peptide bonds. Enzymes which belong to this family, with a diverse array of metabolic and regulatory functions, play critical roles in cell physiology and pathology. ءClp̕s are a class of ATP dependent serine proteases which are composed of a protease (ClpP) and an ATPase (ClpA or ClpX) component. Their involvements in degrading proteins are especially implicated und...
Construction of various fusion proteins of recombinant citrate synthase from thermoplasma volcanium Özdoğan, Seda; Kocabıyık, Semra; Department of Biology (2004) In this study, a strategy called gene splicing by overlap extension, 3Gene SOEing4, was used for the construction of the fusion proteins with the purpose of increasing the thermostability of mesophilic enzymes by incorporation of stability domain from a thermostable enzyme. Gene SOEing is a PCR-based approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. In fusion constructs, as th...
Biochemical characterization of recombinant 20s proteasome from thermoplasma volcanium and cloning of it's regulatory subunit gene Baydar, Gözde; Kocabıyık, Semra; Department of Biotechnology (2006) 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...
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...
The genetic basis of malathion resistance in housefly (Musca domestica L.) strains from Turkey Taskin, V; Kence, M (Springer Science and Business Media LLC, 2004-11-01) Organophosphate insecticide (parathion/diazinon) resistance in housefly ( Musca domestica L.) is associated with the change in carboxylesterase activity. The product of MdalphaE7 gene is probably playing a role in detoxification of xenobiotic esters. In our research, we have isolated, cloned and sequenced the MdalphaE7 gene from five different Turkish housefly strains. High doses of malathion ( 600 mug/fly) were applied in a laboratory environment for one year to Ceyhan1, Ceyhan2, Adana, and Ankara strains ...

Citation Formats

B. Koyuncu, “Expression of recombinant acid protease (thermopsin) gene from Thermoplasma volcanium,” M.S. - Master of Science, Middle East Technical University, 2006.