Exchanging regions of structurally similar proteins to create kimeras allows us to investigate the importance of regions responsible for biological functions of the molecule to be studied. Region exchanges predoff the general secondary structure of the protein so that region's important for it's overall structure can be distinguished from those directly mediating biological functions. It can be turning into select which exact regions to be replaced.
Starting with larger exchanges is usually helpful to identify the protein's key functionality. To begin the procedure, select a suitable protein as a donor to exchange regions with the protein of interest as detailed in the text protocol. Then, obtain the protein amino acid sequences of the recipient and donor proteins from the reference sequence database by first accessing the gene section in the ref seq webpage.
Type the name of the protein of interest in the search box and click search. Click on the gene name for the desired species in the resulting list. Scroll down to the ref seq section to see all of the documented isoforms.
Click on the sequence identifier for the isoform of interest. Scroll down and click on cds to highlight the protein coding region of the gene. On the bottom right of the screen, click on FAFSDA and copy the gene sequence.
Save the DNA sequence using a suitable DNA editing software. When using the freely available APE, open the program, paste the copied sequence in the blank box, select the sequence name, and click save. Now, choose the protein regions to be substituted in the different kimeric constructs by first dividing the protein sequence of interest in distinct structural regions.
To do so, download the structural data of the protein of interest from the PDBe website. Access the PDBe page for the protein and download the PDBe file by clicking download at the right side of the screen. Open the PDBe file in a molecular visualization system, like Pymol.
In Pymol, display the nucleotide sequence, hide the default structural data and select the cartoon view to clearly visualize the protein's structural features. Click on the nucleotide sequence at the top of the screen to highlight different parts of the molecule, noting the amino acids corresponding to each distinctive structural feature. Now, annotate the distinct structural regions on the DNA sequence in APE by opening the DNA sequence, selecting it, and clicking ORFs translate, then click on the last amino acid of the first region to highlight its position in the DNA sequence and select the nucleotide's coding for that region.
Right click on the selection and select new feature to give it a name and a color. Repeat the process for each structural region identified in the previous step. To align the residue sequences of the two proteins, first, obtain the full amino acid sequences of donor and receptor proteins in APE.
As before, open the donor DNA sequence, select it, and click ORFs translate. Then, access the Clustal Omega webpage and then put the amino acid sequences of the two proteins. Each sequence should be proceeded by a text line with protein name to be properly identified.
Scroll down and click submit. Retrieve the alignment file by clicking the download alignment file tab and save it. This file can be opened by any text editing program.
Using the alignment file as a reference, annotate the corresponding structural regions of the donor protein in their DNA sequence. Create a copy of the annotated DNA sequence of the receptor protein and rename it as a kimeric protein. Open the renamed DNA sequence in APE.
Select the region to be exchanged in the donor protein, then select the corresponding region in the renamed receptor protein. Paste the donor protein DNA sequence, and then save the changes. Design the terminal primers using a DNA editor such as APE.
Create a new DNA file, initiate the end terminal primer with a leader sequence. Followed by the first restriction site selected in the vectors MCS and the optional spacer in the initial 18 to 27 base pairs of the gene of interest. In the new DNA file, design the C terminal primer sequence to start with the final 18 to 27 base pairs of the gene of interest followed by an optional spacer, the second restriction site chosen, and the leader sequence.
Highlight the whole sequence, right click, and select reverse compliment to obtain the reverse primer. Now, design primers for each of the border regions in the kimeric constructs. In the DNA sequence of the kimera, highlight a 30 base pair region in the zone where the original and inserted sequences are in contact.
This is comprised of 15 base pairs in each sequence. Copy the region, and paste it in a new DNA file. This sequence will be the forward primer.
Make a copy of the forward primer generated in the previous step, and rename it as the reverse primer. Highlight the primer sequence, right click, and select reverse compliment to generate the reverse primer sequence. Repeat these steps for each contact zone in the kimeric DNA sequence.
Generally, two sets of forward reverse primers are required to generate one kimera, unless the replacement occurs at the N terminal or C terminal regions. Prepare an individual PCR reaction mixture for each of the fragments composing the kimeric protein. First set a one point five mililiter microfuse tube on ice and pipet the different reagents of the PCR mixtures in the order listed in the text protocol.
Ensure correct primers and templates are employed for each PCR reaction. Label two thin walled zero point two mililiter PCR tubes for each reaction and transfer 20 microliters of the corresponding PCR mixture in each tube. Transfer the PCR tubes into a PCR thermocycler and initiate the protocol as detailed in the text protocol.
After the PCR reaction is completed, add four microliters of six x DNA loading buffer in each tube. Insert the one percent agaross gel in an electrophoresis unit and cover with TAE buffer. Carefully load the samples into the gel along with a molecular weight latter.
After running the gel at 80 to 120 volts for 20 to 45 minutes, turn off the electrophoresis unit and remove the agoross gel. Visualize the amplified DNA bands under UV light. Using a razor blade, cut out the individual DNA fragments from the gel and transfer them to labeled two mililiter microfuse tubes.
After using a PCR clean up kit to purify the DNA fragments, quantify the amount of DNA recovered by measuring the absorbance of the samples at 260 nanometers and 340 nanometers in a spectrofetometer. Now, perform PCR amplification to generate the kimeric DNA sequence. First, set up 50 microliters of a PCR reaction to fuse the separate constituents of the kimera as before.
Employ the N terminal and C terminal primers along with 10 nanograms of each of the amplified DNA fragments. Recover and quantify the purified DNA fragment as before, in 30 microliters of nucleus free water. This fragment contains the kimeric DNA sequence flanked by the restriction sites included in the terminal primers.
Generation of a kimeric protein is exemplified with two members of the inter luke and six sytokine family. Oncostaten M in leukemia inhibitory factor. The OSM lift kimera results from exchanging the BC loop region of OSM with a corresponding lift sequence.
The first PCR amplification step consisted of three separate reactions. The N-terminal OSM fragment, which required N-terminal OSM forward and BC start reverse primers used OSM as the template. The LIF BC loop was obtained through BC start forward and BC N reverse primers using LIF as the template.
The C-terminal OSM fragment used BC end forward and C terminal OSM reverse primers as well as OSM as the template. These purified fragments were then used as the template in the second PCR reaction along with N-terminal OSM forward and C-terminal OSM reverse primers to amplify the corresponding OSM LIF BC loop gene sequence. Following purification and transformation into e-coli, individual plasmids were isolated and screened by restriction enzyme digestion for proper insertion of the DNA fragment.
Gel electrophoresis revealed positive hits that were then sent for sequence verification. Kimeric proteins have to be produced and the activity measured in a perfect factionality systems. In order to determine the importance of the replaced regions for that particular faction.
This technique has been very useful and frequently applied, interfered of signally receptors in order to identify key function domains and receptor recognition sites. The number I use for DNA electrophoresis should be handled by using disposable gloves, a laboratory coat and protective eyewear.
