In vitro translation with [34S]-labeled methionine, selenomethionine, and telluromethionine.
Heteroisotope and heteroatom tagging with [(34)S]-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 [(34)S]-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. [figure: see text] TeMet was less incorporated into GFP than Met and SeMet due to its instability in WGE.
Ogra
Yasumitsu
Y
Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba, 260-8675, Japan. ogra@p.chiba-u.ac.jp
Kitaguchi
Takashi
T
Suzuki
Noriyuki
N
Suzuki
Kazuo T
KT
D002851:Chromatography, High Pressure Liquid; D054637:Dual-Specificity Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D008715:Methionine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010936:Plant Extracts / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018036:Selenium Compounds / Q000032:analysis / Q000737:chemistry*; D013460:Sulfur Isotopes / Q000737:chemistry; D013691:Tellurium / Q000032:analysis / Q000737:chemistry*; D014908:Triticum / Q000737:chemistry
D010936:Plant Extracts; D011994:Recombinant Proteins; D018036:Selenium Compounds; D013460:Sulfur Isotopes; D008715:Methionine; D054639:Mitogen-Activated Protein Kinase Phosphatases; D054637:Dual-Specificity Phosphatases; D013691:Tellurium