Luigi Alvarado - Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway

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  Publication Details (including relevant citation   information):

  Arthur, P.K.; Alvarado, L.J., Dayie, T.K. Protein Expression and   Purification 76 (2011) 229-237.


  RNAs, more than ever before, are increasingly viewed as   biomolecules of the future, in the versatility of their functions   and intricate three-dimensional folding. To effectively study   them by nuclear magnetic resonance (NMR) spectroscopy, structural   biologists need to tackle two critical challenges of spectral   overcrowding and fast signal decay for large RNAs. Stable-isotope   nucleotide labeling is one attractive solution to the overlap   problem. Hence, developing effective methods for nucleotide   labeling is highly desirable. In this work, we have developed a   facile and streamlined source of recombinant enzymes from the   pentose phosphate pathway for making such labeled nucleotides.   The Escherichia coli (E. coli) genes encoding ribokinase (RK),   adenine phosphoribosyltransferase (APRT), xanthine/guanine   phosphoribosyltransferase (XGPRT), and uracil   phosphoribosyltransferase (UPRT) were sub-cloned into pET15b   vectors. All four constructs together with cytidine triphosphate   synthetase (CTPS) and human phosphoribosyl pyrophosphate   synthetase isoform 1 (PRPPS) were transformed into the E. coli   BL21(AI) strain for protein over-expression. The enzyme   preparations were purified to >90% homogeneity by a one-step   Ni–NTA

  affinity chromatography, without the need of a further   size-exclusion chromatography step. We obtained yields of 1530,   22, 482, 3120, 2120 and 2280 units of activity per liter of   culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively;   the specific activities were found to be 70, 22, 21, 128, 144 and   113 U/mg, respectively. These specific activities of these enzyme   constructs are comparable to or higher than those previously   reported. In addition, both the growth conditions and   purification protocols have been streamlined so that all the   recombinant proteins can be expressed, purified and characterized   in at most 2 days. The availability and reliability of these   constructs should make production of fully and sitespecific   labeled nucleotides for making labeled RNA accessible and   straightforward, to facilitate highresolution NMR spectroscopic   and other biophysical studies.

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