Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins

Abstract

Source: Danis, C. et. al., Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins. J. Vis. Exp. (2016).

This video demonstrates the use of nuclear magnetic resonance spectroscopy (NMR) techniques to identify multiple phosphorylations in a protein. The phosphorylation of a protein at specific amino acid causes the deshielding of the neighboring amide hydrogen, which generates the spectral difference.

Protocol

1. Production of ¹⁵N, ¹³C-Tau (Figure 1)

1. ATransform pET15b-Tau recombinant T7 expression plasmid into BL21(DE3) competent Escherichia coli bacterial cells.
   NOTE: the cDNA coding for the longest (441 amino acid residues) Tau isoform is cloned between NcoI and XhoI restriction sites in the pET15b plasmid.
   1. Mix gently 50 µl of competent BL21(DE3) cells, forming 1-5 x 10⁷ colonies per µg of plasmid DNA, with 100 ng of plasmid DNA in a 1.5 ml plastic tube.
      NOTE: Codon-usage optimized bacterial strains for eukaryotic ....

Results

Figure 1: Scheme of the main steps of recombinant protein production and isotopic labeling. Steps from bacteria transformation to recombinant protein production are outlined as described in paragraph 1 of the protocol.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pET15B recombinant T7 expression plasmid	Novagen	69257	Keep at -20°C
BL21(DE3) transformation-competent E.coli bacteria	New England Biolabs	C2527I	Keep at -80°C
Autoclaved LB Broth, Lennox	DIFCO	240210	Bacterial Growth Medium
MEM vitamin complements 100X	Sigma	58970C	Bacterial Growth Medium Supplement
<sup>15</sup>N, <sup>13</sup>C-ISOGRO complete medium powder	Sigma	608297	Bacterial Growth Medium Supplement
<sup>15</sup>NH4Cl	Sigma	299251	Isotope
<sup>13</sup>C-Glucose	Sigma	389374	Isotope Keep at 4&#176;C
Protease inhibitor tablets	Roche	5056489001	1 tablet in 1ml is 40X solution that can be kept at -20&#176;C
DNaseI	EUROMEDEX	1307	Keep at -20&#176;C
Homogenizer (EmulsiFlex-C3)	AVESTIN		Lysis is realized at 4&#176;C
Pierce<sup>TM</sup> Unstained Protein MW Marker	Pierce	266109
Active human MEK1 kinase, GST-Tagged	Sigma	M8822	Keep at -80&#176;C
AKT&#196; Pure chromatography system	GE Healthcare		FPLC
HiTrap SP Sepharose FF (5 mL column)	GE Healthcare	17-5156-01	Cation exchange chromatography columns
HiPrep 26/10 Desalting	GE Healthcare	17-5087-01	Protein Desalting column
PD MidiTrap G-25	GE Healthcare	28-9180-08	Protein Desalting column
Tris D11, 97% D	Cortecnet	CD4035P5	Deuterated NMR buffer
5 mm Symmetrical Microtube SHIGEMI D<sub>2</sub>O ( set of 5 inner &#38; outerpipe)	Euriso-top	BMS-005B	NMR Shigemi Tubes
eVol kit-electronic syringe starter kit	Cortecnet	2910000	Pipetting
Bruker 900MHz AvanceIII with a triple resonance cryogenic probehead	Bruker		NMR spectrometer for data acquisition
Bruker 600MHz DMX600 with a triple resonance cryogenic probehead	Bruker		NMR spectrometer for data acquisition