Heteroisotope and heteroatom tagging with [34S]-enriched methionine (Met), selenomethionine (SeMet), and telluromethionine (TeMet) was applied to in vitro translation. Green fluorescent protein (GFP) and JNK stimulatory phosphatase-1 (JSP-1) genes were translated with wheat germ extract (WGE) in the presence of Met derivatives. GFPs containing Met derivatives were subjected to HPLC coupled with treble detection, i.e., a photodiode array detector, a fluorescence detector, and an inductively coupled plasma mass spectrometer (ICP-MS). The activities of JSP-1-containing Met derivatives were also measured. GFP and JSP-1 containing [34S]-Met and SeMet showed comparable fluorescence intensities and enzyme activities to those containing naturally occurring Met. TeMet was unstable and decomposed in WGE, whereas SeMet was stable throughout the experimental period. Thus, although Te was the most sensitive to ICP-MS detection among S, Se, and Te, TeMet was less incorporated into the proteins than Met and SeMet. Finally, the potential of heteroisotope and heteroatom tagging of desired proteins in in vitro translation followed by ICP-MS detection was discussed.
ICP-MS Stable isotope Methionine Selenomethionine Telluromethionine GFP In vitro translation
We would like to acknowledge Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Nos. 16689005, 16209004, and 19390033), and the financial support from Agilent Technologies Foundation, USA. We also wish to thank ZOEGENE Co. for the in vitro translation kit and control mRNA, and Showa Denko for the HPLC system.