An economical method for (15)N/(13)C isotopic labeling of proteins expressed in Pichia pastoris.
An economical method for (15)N/(13)C isotopic labeling of proteins expressed in Pichia pastoris.
复制标题
一种对毕赤酵母中表达的蛋白质进行 (15)N/(13)C 同位素标记的经济方法。
DOI：
10.1093/oxfordjournals.jbchem.a002957
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发表时间：
2001
影响因子：
2.7
通讯作者：
Krishna,NR
中科院分区：
文献类型：
--
作者：
Rodriguez,E;Krishna,NR
We report a new and cost-effective approach to prepare15N/13C labeled proteins for NMR using thePichia pastorisexpression system. Four protocols (P1 to P4) were defined and compared using recombinant Ovine interferon-tau (rOvIFN-τ). Our results demonstrate that in order to get full incorporation of15N and13C, the isotopes are not totally required during the initial growth phase of P. pastoris culture. The addition of small amounts of15N and13C compounds 6 h prior to the methanol induction phase is sufficient to obtain 99% incorporation of heavy isotopes into the protein. Our optimized protocol P4 is two-thirds less costly than the classical method using15N and13C isotopes during the entire growth phase.
