Published: November 30th, 2018
In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of ryanodine receptor from diamondback moth (Plutella xylostella).
Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 Å, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.
Ryanodine receptors (RyRs) are specific ion channels, which mediate the permeation of Ca2+ ions across the sarcoplasmic reticulum (SR) membranes in muscle cells. Therefore, they play an important role in the excitation contraction coupling process. In its functional form, RyR assembles as a homo-tetramer with a molecular mass of >2 MDa, with each subunit comprising of ~5000 amino acid residues. In mammals, there are three isoforms: RyR1- skeletal muscle type, RyR2- cardiac muscle type and RyR3- ubiquitously expressed in different tissues1.
In insects there is only one type of RyR, which is expressed in....
1. Gene Cloning, Protein Expression, and Purification
The N-terminal domain of DBM RyR was expressed as a fusion protein with a hexahistidine tag, a MBP tag and a TEV protease cleavage site. We followed a five-step purification strategy to obtain a highly pure protein, suitable for crystallization purpose. At first, the fusion protein was purified from the soluble fraction of cell lysate by Ni-NTA column (HisTrap HP). Next, the fusion protein was subjected to TEV pro.......
In this paper, we describe the procedure to recombinantly express, purify, crystallize and determine the structure of DBM RyR NTD. For crystallization, a crucial requirement is to obtain proteins with high solubility, purity and homogeneity. In our protocol, we chose to use pET-28a-HMT vector as it contains a hexahistidine tag and MBP tag, both of which could be utilized for purification to obtain a higher fold purity. Additionally, the MBP tag aids in the solubility of the target protein. We purified the protein by five.......
Funding for this research was provided by: National Key Research and Development Program of China (2017YFD0201400, 2017YFD0201403), National Nature Science Foundation of China (31320103922, 31230061), and Project of National Basic Research (973) Program of China (2015CB856500, 2015CB856504). We are grateful to the staff on the beamline BL17U1 at Shanghai Synchrotron Radiation Facility (SSRF).....
|This modified pET vector contains a hexahistidine tag, an MBP fusion protein and a TEV protease cleavage site at the N-terminus (Lobo and Van Petegem, 2009)
|E. coli BL21 (DE3) strain
|HisTrapHP column (5 mL)
|Amylose resin column
|New England Biolabs
|Q Sepharose high-performance column
|Amicon concentrators (10 kDa MWCO)
|Superdex 200 26/600 gel-filtration column
|Automated liquid handling robotic system
|Art Robbins Instruments
|96 Well CrystalQuick
|Mounted CryoLoop - 20 micron
|Puck dewar loading tool
|Plasmid mini-prep kit
|Gel extraction kit
|SspI restriction endonuclease
|T4 DNA polymerase
|Sorvall LYNX 6000
|Protein purification system
|IzIt crystal dye
|Index crystal screen
|Structure crystal screen
|ProPlex crystal screen
|PACT premier crystal screen
|JCSG-plus crystal screen
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