Cost-effective isotope labeling technique developed for ^(15)N/^(13)C-labeled proteins
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A newly developed cost-effective approach to prepare ^(15)N/^(13)C-labeled protein for NMR studies is presented. This method has been successfully applied to isotopically labeling of PTK6 SH2 domain and MTH 1880 protein. The production method generates cell density using a growing media containing ^(15)NH_(4)Cl, ^(12)C_(6)-D-glucose. Following a doubling time period for unlabeled metabolite exhaustion and then addition ^(13)C_(6)-D-glucose into a M9 growing media, the cells are induced. Our results demonstrate that in order to get full incorporation of ^(13)C, the isotopes are not totally required during the initial growth phase before induction. The addition of small amounts of ^(13)C_(6)-D-glucose to the induction phase is sufficient to obtain more than 95% incorporation of isotopes into the protein. Our optimized protocol is two-thirds less costly than the classical method using ^(13)C isotope during the entire growth phase.
Abstract
A newly developed cost-effective approach to prepare ^(15)N/^(13)C-labeled protein for NMR studies is presented. This method has been successfully applied to isotopically labeling of PTK6 SH2 domain and MTH 1880 protein. The production method generates cell density using a growing media containing ^(15)NH_(4)Cl, ^(12)C_(6)-D-glucose. Following a doubling time period for unlabeled metabolite exhaustion and then addition ^(13)C_(6)-D-glucose into a M9 growing media, the cells are induced. Our results demonstrate that in order to get full incorporation of ^(13)C, the isotopes are not totally required during the initial growth phase before induction. The addition of small amounts of ^(13)C_(6)-D-glucose to the induction phase is sufficient to obtain more than 95% incorporation of isotopes into the protein. Our optimized protocol is two-thirds less costly than the classical method using ^(13)C isotope during the entire growth phase.
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