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In vitro stability and expression of green fluorescent protein under high pressure conditions
M A Ehrmann1, C H Scheyhing, R F Vogel
1Lehrstuhl für Technische Mikrobiologie, Technische Universität München, Freising-Weihenstephan, Germany. M.ehrmann@lrz.tu-muenchen.de
Aims:
The objective of this work was to evaluate the use of wild-type GFP and mutant forms thereof as reporter for gene expression under high pressure conditions.
Methods And Results:
The intensity of fluorescence after high pressure treatment was checked by subjecting cells, crude protein extracts containing GFPs and purified GFPs to pressures ranging from 100 MPa to 900 MPa. All tested GFP's retained fluorescence up to 600 MPa without loss of intensity. Expression of GFP under sublethal conditions was investigated in Escherichia coli with plasmid pQBI63, in which rsGFP is placed downstream of the T7 RNA polymerase binding site. T7 RNA polymerase is controlled in E. coli BL21 (DE3) pLysS by an IPTG inducible lacUV5 promoter. A pressure induced increase of GFP expression was monitored at 50 Mpa and 70 MPa.
Conclusion:
Fluorescence of GFPs is not influenced at pressures at which protein expression still occurs. We showed that the expression system used is inducible by pressurized conditions.
Significance And Impact Of The Study:
This study proved GFP to be a suitable reporter for gene expression studies capable to detect pressure induced gene expression.