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 TP1&rsquo / /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 Anti-His HRP conjugates which are specific for 6xHis tag, and DAB chromogenic substrate was used for colony blot procedure.
PCR amplified thermopsin gene containing 3080bp could not expressed in pQE30 and 31 vectors in TG1 strains. It is thought that pQE32 open reading frame can be true for thermopsin gene (3080bp).
Three expression constructs, pQE31-1, pQE31-4 and pQE31-6 plasmids containing PCR amplified 3070bp thermopsin gene were confirmed as true recombinant plasmids according to both colony blot hybridization result and restriction digestion profile the agarose gel.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/3/12606995/index.pdf |
| Date | 01 January 2006 |
| Creators | Koyuncu, Bilsev |
| Contributors | Kocabiyik, Semra |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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