Patent Application
20180371437
In general, the present invention relates to the creation of crystals from acetylcholinesterase and to systems that utilize such crystals to produce digital models through X-ray crystallography. More particularly, the present invention relates to the methodology of creating crystals from insect acetylcholinesterase.
Acetylcholinesterase, also known as AChE is a serine protease that hydrolyzes the neurotransmitter acetylcholine (Ach). AChE is found at mainly neuromuscular junctions and cholinergic brain synapses, where it serves to terminate synaptic transmission. For a cholinergic neuron to receive another impulse, ACh must be released from the ACh receptor. This occurs only when the concentration of ACh in the synaptic cleft is very low.
During neurotransmission, ACh is released from the nerve into the synaptic cleft and binds to ACh receptors on the post-synaptic membrane, therein relaying the signal from the nerve. AChE, which is also located on the post-synaptic membrane, terminates the signal transmission by hydrolyzing the ACh, therein liberating a choline. The liberated choline is taken up again by the pre-synaptic nerve and ACh is synthesized by combining with acetyl-CoA through the action of choline acetyltransferase.
Inhibition of AChE leads to accumulation of ACh in the synaptic cleft. This results in impeded neurotransmission or a cessation of neurotransmission. Consequently, inhibition of AChE may lead to death. As a result, inhibitors of AChE have proven to be very effective nerve toxins and insecticides. Therefore, by studying compounds that inhibit AChE in various insects, a pathway for studying insecticides may be found that is useful in the targeted control of pest insects, such as the species of mosquito that carries malaria.
In use, the residual spraying of anticholinesterase insecticides has been useful in controlling insects, such as the mosquitos that spread malaria. However, widespread application of anticholinesterase insecticides has led to mutations and the rise of insecticide-resistant insect strains. In mosquitos, common insecticide-resistant mosquito strains include a G280S mutation, which is sometimes referred to as a G119S mutation. This mutation affects enzyme acetylcholinesterase in the insect nervous system, therein inhibiting the effects of the insecticide.
To the best of the Applicant's knowledge, there are no structures of mosquito AgAChE that are available that include the G280S mutation and that are useful for X-ray crystallography. In the prior art, the Applicant has previously developed a system and method of obtaining high-resolution crystal structures of human AChE. Such a system and method are disclosed in co-pending U.S. patent application Ser. No. 15/469,227. However, the application to mutated insect AChE remain undisclosed.
In order to effectively study the effects of any compound that reacts with insect AChE, the insect AChE must first be accurately modeled. The way that insect AChE is modeled requires that crystals of insect AChE be formed. The crystals are then subjected to X-ray crystallography. X-ray crystallography is a method used for determining the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional model of the density of electrons within the crystal. From this study of electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder, and various other information that can be used to create an accurate digital model.
A need therefore exists for a system and method of creating better crystals of insect AChE, therein resulting in better modeling using X-ray crystallography. This need is met by the present invention as described below.
The present invention is a method of creating crystals of insect acetylcholinesterase, such as mosquito acetylcholinesterase. The method includes obtaining a first polynucleotide that encodes for acetylcholinesterase in a targeted insect. The first polynucleotide contains a targeted catalytic core sequence that is required for biological functioning and is constant across different cDNA sources for the insect species. The targeted catalytic core sequence has a known sequence of bases between a specific first codon and a stop codon.
A recombinant DNA construct is formed by adding a fusion protein and a polyhistidine tag to the targeted catalytic core sequence prior to the specified first codon. The recombinant DNA construct is amplified. Additionally, the recombinant DNA construct can be further modified by adding known mutations that cause resistance to acetylcholinesterase inhibitor insecticides.
Bacterial cell colonies on a growth medium are transfected with the recombinant DNA construct. The cell colonies package the DNA construct into a larger DNA construct that can be isolated. This larger DNA construct which contains the recombinant DNA can be used to produce a virus in insect cells which causes them to secrete a polypeptide encoded by contained recombinant DNA construct into the growth medium. The polypeptide includes the polyhistidine tag and part of the targeted catalytic core sequence between the polyhistidine tag and the stop codon.
The polypeptide encoded by the recombinant DNA construct is separated from the growth medium to form a concentrate. The polyhistidine tag is removed from the concentrate. The concentrate is exchanged into a buffer to create a buffered concentrate. Crystals, suitable for X-ray crystallography are then grown with the buffered concentrate.
For a better understanding of the present invention, reference is made to the following description of an exemplary embodiment thereof, considered in conjunction with the accompanying drawings, in which:
The present invention methodology can be used to model insect acetylcholinesterase (AChE). The methodology is especially adapted for modeling insect AChE that contains mutations that make the insect resistant to AChE inhibitor insecticides. Although the methodology can be used to model AChE for a variety of insects, such as agricultural pests, the present invention methodology is particularly useful in modeling disease carrying insects, such as mosquitos. Accordingly, in describing the present invention methodology, its application to mosquito AChE is used as the exemplary embodiment. Mosquitos have known genetic mutations that make some mosquitos resistant to insecticides and therefore provide one of the best examples for describing the methodology. The methodology described and illustrated is exemplary and can be varied using undescribed, yet functionally equivalent process steps. The methodology described, however, is merely exemplary and should not be considered a limitation to the novelty of the invention as described.
Referring to
Referring to
To create the recombinant DNA construct 20, the targeted catalytic core sequence 12 that has been isolated is altered. A DNA coding sequences for a yeast SUMOstar fusion protein 22 is fused to the front end of the targeted catalytic core sequence 12. This is accomplished using molecular biology techniques, such as overlap extension polymerase chain reaction (PCR) protocols to enhance protein expression and secretion of complex proteins. This corresponds to a position prior to codon 162 of the initial ACE-1 gene cDNA fragment 10. See Block 24. The recombinant fusion also provides a secretion signal 26 that later directs the secretion of produced proteins into a cell growth media. See Block 28.
A TEV-protease cleavable polyhistidine tag 30 is added to the recombinant DNA construct 20. See Block 32. The TEV-protease cleavable polyhistidine tag 32 is inserted between the yeast SUMOstar fusion protein 22 and the first codon of the targeted catalytic core sequence 12 at codon 162 (GAC) of the initial ACE-1 gene cDNA fragment 10. See
One or more selected mutations can be added to the recombinant DNA construct 20. In the illustrated example, a G280S mutation 34 is inserted into the targeted catalytic core sequence 12 after codon 280 of the initial ACE-1 gene cDNA fragment 10. The mutation is added using a commercial baculovirus expression system, such as the Invitrogen™ Bac to Bac™ brand baculovirus expression system sold by Life Technology Corporation. The baculovirus expression system contains a baculovirus shuttle vector. A selected mutation is introduced into the baculovirus shuttle vector that produces a specific mutation, such as a G280S insecticide-resistant mutation. See Block 36. It will be understood that the illustrated introduction of a G280S insecticide-resistant mutation is exemplary. Other known mutations that effect insecticide resistance can also be used. The bacterial strain for the selected mutation is transformed with the baculovirus shuttle vector and colonies are screened for recombination events which cause the bacteria to produce baculovirus DNA (bacmid). See Block 38. The baculovirus DNA is screened and colonies grow. See Block 40 and Block 42.
A growth medium of insect cells is prepared and transfected with the baculovirus DNA. An initial virus is produced and used to infect larger cultures of insect cells. This amplifies the virus. See Block 44. A final culture of insect cells in a cell growth medium are infected by the amplified virus. See Block 46. Due to the secretion signal 26 and SUMOstar fusion protein 22 present in the recombinant DNA construct 20, the recombinant AgAChE G280S mutant fusion protein is secreted into the cell growth medium for harvesting. See Block 48. After secretion, the cells are removed and what is left is the remnants of the cell growth medium that contains the secreted recombinant AChE G280S fusion protein. That is, the cell colonies secrete the segment of the recombinant DNA construct 20 that corresponds from the polyhistidine tag 30 before base position 162 to stop codon TAG just beyond base position 574. The secreted recombinant AChE G280S fusion protein is still tagged with the polyhistidine tag 30. The remnants of the cell growth medium contacting the secreted recombinant AgAChE G280S fusion protein is collected. See Block 50.
Referring to
The purified AgAChE G280S protein is dialyzed overnight into a storage buffer, such as 10 mM HEPES (pH 7.0) and 10 mM NaCl. The solution is concentrated to 5 mg/ml for crystallization. See Block 60.
Crystals of the purified AgAChE G280S protein are grown by sitting drop vapor diffusion at 4° C. against the crystallization buffer. See Block 62. Hexagonal rod-shaped crystals (20 μm×20 μm×300 μm) are typically nucleated within 14 days and grow to full size over 60 days. Once the crystals are full size, they are harvested. The crystals can be used directly, but are preferably soaked in a ligand, prior to harvesting. See Block 64. Alternatively, a ligand can be mixed with the crystallization buffer. The harvested crystals can then be cut and subjected to X-ray crystallography and modeling in the traditional manner. See Block 66 and Block 68.
It will be understood that the method steps of the present invention that are illustrated and described are merely exemplary and that a person skilled in the art can make many variations to those method steps. All such embodiments are intended to be included within the scope of the present invention as defined by the appended claims.
This application claims the benefit of U.S. Provisional Patent Application No. 62/524,728, filed Jun. 26, 2017.
| Number | Date | Country | |
|---|---|---|---|
| 62524728 | Jun 2017 | US |
