RESUMEN
To achieve the production of a thermostable DNA polymerase free from bacterial DNA contamination, we developed eukaryote-made thermostable DNA (Taq) polymerase. The novel eukaryote-made thermostable DNA polymerase resolves the problem of contaminating bacterial DNA in conventional bacterially made thermostable DNA polymerase as a result of its manufacture and incomplete purification. Using eukaryote-made thermostable DNA polymerase, the sensitive and reliable detection of bacteria becomes feasible for large fields, thereby making the development of a wide range of powerful applications possible.
Asunto(s)
Biotecnología/métodos , ADN Bacteriano/análisis , Polimerasa Taq/química , Polimerasa Taq/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Polimerasa Taq/biosíntesis , Polimerasa Taq/genéticaRESUMEN
We have developed a new expression vector, pcI(ts) ind(+), based upon the powerful rightward promoter of bacteriophage lambda, which is controlled by a temperature-sensitive and chemically-inducible version of the lambda repressor on the same plasmid. Locating the repressor gene on the plasmid makes this vector "portable" in that it can be used to transform any strain of Escherichia coli. Hence, control over strains, induction conditions, and harvest times can be used to optimize yields of heterologous proteins. To provide a proof of concept, we show that E. coli recA(+) and recA(-) host cells transformed with pcI(ts) ind(+) modKlenTaq1 (a modified version of the large fragment of Thermus aquaticus DNA polymerase I) could be grown to high cell densities in multiple shake-flasks. A mutant version of modKlenTaq1 (V649C) could be induced by simply raising the thermostat setting from 30 to 37 degrees C and (in the case of recA(+) cells) adding nalidixic acid to achieve full induction (12-13% of the total cellular protein). Using a rapid, two-step purification process, it was possible to purify nearly 300 mg of modKlenTaq1 V649C from six 2.8-L baffle-bottomed shake-flasks each holding 1.5L of culture for a final yield of approximately 33 mg per liter or 3mg of purified enzyme per gram of cells wet weight.
Asunto(s)
Técnicas de Cultivo de Célula , ADN Polimerasa I/biosíntesis , ADN Polimerasa I/aislamiento & purificación , Plásmidos/metabolismo , Polimerasa Taq/biosíntesis , Polimerasa Taq/aislamiento & purificación , Clonación Molecular , Escherichia coli/genética , Vectores Genéticos , Plásmidos/efectos de los fármacos , Plásmidos/genética , Thermus/enzimologíaRESUMEN
The Thermus aquaticus DNA Polymerase I (Taq Pol I) gene was cloned into the pOSEX4 plasmid under the osmo-inducible promoter proU and subsequently expressed into the Escherichia coli MKH13 strain. The suitability of the enzyme in polymerase assays was determined in standard 35S dATP incorporation tests and by PCR. The Taq Pol I expression in this system, which is under the control of the osmotic pressure in the growth medium, was analyzed in different media and in different sodium chloride concentrations. A study of the osmolarity effects in the growth of the strain and in Taq Pol I expression shows that an increase in sodium chloride concentration limits the growth. At 0.25 M of NaCl maximum activity was observed; at higher values of osmolarity, we found an unexpected decline of activity. This is the first report of using the pOSEX vector for the expression of an heterologous protein and it is very advantageous to make a regulated, non toxic, simple and cost-effective manner of induction in a biotechnology process using just NaCl or other non-permeable osmolyte.
Asunto(s)
Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica , Ósmosis , Polimerasa Taq/genética , Biotecnología , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Microbiología Industrial , Concentración Osmolar , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Solución Salina Hipertónica , Polimerasa Taq/biosíntesisRESUMEN
Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method. Taq DNA polymerase has a domain at the amino terminus (residue 1 to 290) that has a 5' exonuclease activity and a domain at the C-terminus that catalyzes polymerase reaction. Taq DNA polymerase is classified into the pol I family which is represented by E. coli DNA polymerase I. The alignment of amino acid sequences for the 5' exonuclease domains of the pol I family DNA polymerases shows six highly conserved sequences called motifs A to F. Motif C contains three positively charged residues such as 74Arg, 82Lys and 85Arg which might be involved in catalysis. In order to understand the function of those residues, they are mutagenized to alanine. The 5' exonucleolytic activities of those mutated 5' exonucleases decreased by 80 to 90%, thereby implying that three positively charged residues play certain roles in the 5' exonuclease catalysis.