In vitro stability and expression of green fluorescent protein under high pressure conditions

Aims: The objective of this work was to evaluate the use of wild-type GFP a nd mutant forms thereof as reporter for gene expression under high pressure conditions. Methods and Results: The intensity of fluorescence after high pressure trea tment was checked by subjecting cells, crude protein extracts containing GF Ps and purified GFPs to pressures ranging from 100 MPa to 900 MPa. All test ed GFP's retained fluorescence up to 600 MPa without loss of intensity. Exp ression of GFP under sublethal conditions was investigated in Escherichia c oli 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 (D E3) pLysS by an IPTG inducible lacUV5 promoter. A pressure induced increase of GFP expression was monitored at 50 Mpa and 70 MPa. Conclusions: Fluorescence of GFPs is not influenced at pressures at which p rotein expression still occurs. We showed that the expression system used i s inducible by pressurized conditions. Significance and Impact of the Study: This study proved GFP to be a suitabl e reporter for gene expression studies capable to detect pressure induced g ene expression.