Electron beam irradiation as protection against the environmental release of recombinant molecules for biomaterials applications.
In biomaterials applications there exists a need to protect against the environmental release of recombinant microorganisms and transmissible genetic material and to prevent the recovery of proprietary genetic information. Irradiation technologies have long been used to eliminate microorganisms associated with spoilage and contamination and recent studies have demonstrated that moderate doses of irradiation may be used to sterilize medically important proteins without causing adverse effects in their desirable biological properties. Recombinant Escherichia coli cells expressing organophosphate hydrolase (OPH, E.C. 3.1.8.1), an important enzyme for the detection and decontamination of neurotoxic pesticides and chemical warfare agents, were subjected to electron beam irradiation to gauge its effect on enzymatic activity, cell viability and DNA recoverability. Bacterial samples were irradiated at 2, 20 and 200 kGy using a 10 MeV electron source. Irradiation levels of 2 to 20 kGy were sufficient to eliminate viable cells without affecting OPH enzymatic activity. Biologically active DNA was recovered via PCR from all samples through the 20 kGy irradiation level. While DNA was not recovered from samples at the 200 kGy exposure level, protein activity was reduced by 19 to 78%, depending on the method of cell preparation. These results demonstrate that irradiation can be effective in preventing the release of recombinant organisms intended for use in biomaterials applications without eliminating enzymatic activity and suggests that further research may indicate specific conditions whereby DNA recovery can be eliminated while retaining sufficient enzymatic activity for targeted biomaterials applications.
Gold
R Shane
RS
Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
Maxim
Joseph
J
Halepaska
Douglas J
DJ
Wales
Melinda E
ME
Johnson
Douglas A
DA
Wild
James R
JR
D043303:Aryldialkylphosphatase / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D002470:Cell Survival / Q000528:radiation effects; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D004583:Electrons; D004777:Environment; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000528:radiation effects; D005818:Genetic Engineering / Q000379:methods*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*
D004274:DNA, Recombinant; D011994:Recombinant Proteins; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases; D043303:Aryldialkylphosphatase