Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway.
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 site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies.
Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742-3360, USA.
Patrick K
PK
Arthur
Luigi J
LJ
Alvarado
T Kwaku
TK
Dayie
D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009532:Nickel / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010427:Pentose Phosphate Pathway; D010430:Pentosyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012268:Ribose-Phosphate Pyrophosphokinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D006639:Histidine; D009532:Nickel; D010430:Pentosyltransferases; C510474:PRPS1 protein, human; D012268:Ribose-Phosphate Pyrophosphokinase; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase