Cabrera AY, Martínez GD, Pérez CER, Márquez PGJ, Feble ML

Language: English
References: 14
Page: 14-18
PDF size: 156.85 Kb.

ABSTRACT

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.

REFERENCES

1. Booth, I.R. & C.F. Higgins. 1990. Enteric bacteria and osmotic stress: intracellular potassium glutamate as a secondary signal of osmotic stress? FEMS. Microb. Rev. 6: 239-246.

2. Csonka, L.N. 1982. A third L-proline permease in Salmonella typhimurium which functions in media of elevated osmotic strength. J. Bacteriol. 151:1433-1443.

3. Cairney, J.; I.R. Booth & C.S. Higgins. 1985. Osmoregulation of gene expression in Salmonella typhimurium: proU encodes an osmotically induced betain transport system. J. Bacteriol. 164:1224-1232.

4. Chien, A.; D.B. Edgar & J.M. Trela. 1976. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus J. Bacteriol. 127:1550-1557.

5. Engelke, D.R.; A. Krikos; M.E. Bruck & D. Ginsburg. 1990. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal. Biochem. 191:396-400.

6. Herbst, B.; S. Kneip & E. Bremer. 1994. pOSEX: vectors for osmoticaly controlled and finely tuned gene expression in Escherichia coli. Gene. 151: 137-142.

7. Lawyer, F.C.; S. Stoffel; R.K. Saiki; K. Myambo; R. Drummond &, D.H. Gelfand. 1989. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. J Biol Chem. 264:6427-6437.

8. Leonart, R; Campos M.; Suárez A.; Vázquez R.; de la Fuente J.; & Herrera L.. 1991. Molecular Cloning of the gene; expression in E. coli and purification of the Thermus aquatic DNA Polymerase I. Acta Biotechnol. 11:67-71.

9. Livingston, D.M.; D.C. Hinkel &, C.C. Richardson. 1975. Deoxyribunucleic acid polymerase III of Escherichia coli. Purification and properties. J. Biol. Chem. 250:461-469.

10. Luncht, J.M. & E. Bremer.1994. Adaptation of Escherichia coli to high osmolarity environments: Osmoregulaton of the high affinity glycine betaine transport system ProU. FEMS Microb. Rev. 14:3-20.

11. Mullis, K.B. & F. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Meth. in Enzymol. 255: 335-350.

12. Saiki, R.K.; S. Scharf; F. Faloona; K.B. Mullis; G.T. Horn; H.A. Erlich & N. Arnehein. 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 230:1350-1354.

13. Sambrook, J.; E.F. Frtsch & T. Maniatis. 1989. Molecular cloning. A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

14. Strom, A.R.; P. Falkenberg &, B. Landfald. 1986. Genetics of osmoregulation in Escherichia coli: uptake and biosynthesis of organic osmolytes.FEMS. Microb. Rev. 39:79-86.