Name: identification of functional protein regions through chimeric protein construction
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Description: Watch this Scientific Journal Video about Identification of Functional Protein Regions Through Chimeric Protein Construction at JoVE.com

This work was supported by the Max Planck Society and the Sch&#252;chtermann-Clinic (Bad Rothenfelde, Germany). Part of this research was originally published in the Journal of Biological Chemistry. Adrian-Segarra, J. M., Schindler, N., Gajawada, P., L&#246;rchner, H., Braun, T. &#38; P&#246;ling, J. The AB loop and D-helix in binding site III of human Oncostatin M (OSM) are required for OSM receptor activation. J. Biol. Chem. 2018; 18:7017-7029. &#169; the Authors.

1. Chimeric Protein Design
<ol>
	<li>Select a suitable protein (donor) to exchange regions with the protein of interest (recipient) The donor protein should be structurally similar, ideally belonging to the same protein family, but lacking the biological activity to be used as readout. If no structurally related proteins are known, potential candidates can be identified using an automated tool such as the Vector Alignment Search Tool (VAST)14,15
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		<li>Acc...

<ol>
	<li>Huising, M. O., Kruiswijk, C. P., Flik, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+and+evolution+of+class-I+helical+cytokines.">Phylogeny and evolution of class-I helical cytokines.</a> The Journal of Endocrinology. 189 (1), 1-25 (2006).</li><li>Brocker, C., Thompson, D., Matsumoto, A., Nebert, D. W., Vasiliou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+divergence+and+functions+of+the+human+interleukin+(IL)+gene+family.">Evolutionary divergence and functions of the human interleukin (IL) gene family.</a> Human Genomics. 5 (1), 30-55 (2010).</li><li>Bravo, J., Heath, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+recognition+by+gp130+cytokines.">Receptor recognition by gp130 cytokines.</a> The EMBO Journal. 19 (11), 2399-2411 (2000).</li><li>Schneider, G., Fechner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-based+de+novo.+design+of+drug-like+molecules.">Computer-based de novo. design of drug-like molecules.</a> Nature Reviews Drug Discovery. 4 (8), 649-663 (2005).</li><li>Heydenreich, F. M., Milju&#353;, T., Jaussi, R., Benoit, R., Mili&#263;, D., Veprintsev, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+mutagenesis+using+a+two-fragment+PCR+approach.">High-throughput mutagenesis using a two-fragment PCR approach.</a> Scientific Reports. 7 (1), 6787 (2017).</li><li>Adrian-Segarra, J. M., Schindler, N., Gajawada, P., L&#246;rchner, H., Braun, T., P&#246;ling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AB+loop+and+D-helix+in+binding+site+III+of+human+Oncostatin+M+(OSM)+are+required+for+OSM+receptor+activation.">The AB loop and D-helix in binding site III of human Oncostatin M (OSM) are required for OSM receptor activation.</a> The Journal of Biological Chemistry. 293 (18), 7017-7029 (2018).</li><li>Wang, Y., Pallen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+wild+type,+truncated,+and+mutant+forms+of+the+intracellular+region+of+the+receptor-like+protein+tyrosine+phosphatase+HPTP+beta.">Expression and characterization of wild type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP beta.</a> The Journal of Biological Chemistry. 267 (23), 16696-16702 (1992).</li><li>Lim, J., Yao, S., Graf, M., Winkler, C., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+analysis+of+full-length+midkine+reveals+novel+residues+important+for+heparin+binding+and+zebrafish+embryogenesis.">Structure-function analysis of full-length midkine reveals novel residues important for heparin binding and zebrafish embryogenesis.</a> The Biochemical Journal. 451 (3), 407-415 (2013).</li><li>Kim, K. -. W., Vallon-Eberhard, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+structure/function+and+expression+analysis+of+the+CX3C+chemokine+fractalkine.">In vivo structure/function and expression analysis of the CX3C chemokine fractalkine.</a> Blood. 118 (22), 156-167 (2011).</li><li>Chollangi, S., Mather, T., Rodgers, K. K., Ash, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+loop+structure+in+oncostatin+M+determines+binding+affinity+toward+oncostatin+M+receptor+and+leukemia+inhibitory+factor+receptor.">A unique loop structure in oncostatin M determines binding affinity toward oncostatin M receptor and leukemia inhibitory factor receptor.</a> The Journal of Biological Chemistry. 287 (39), 32848-32859 (2012).</li><li>Aasland, D., Schuster, B., Gr&#246;tzinger, J., Rose-John, S., Kallen, K. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+leukemia+inhibitory+factor+receptor+functional+domains+by+chimeric+receptors+and+cytokines.">Analysis of the leukemia inhibitory factor receptor functional domains by chimeric receptors and cytokines.</a> Biochemistry. 42 (18), 5244-5252 (2003).</li><li>Hermanns, H. M., Radtke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+leukemia+inhibitory+factor+receptor+and+oncostatin+M+receptor+to+signal+transduction+in+heterodimeric+complexes+with+glycoprotein+130.">Contributions of leukemia inhibitory factor receptor and oncostatin M receptor to signal transduction in heterodimeric complexes with glycoprotein 130.</a> Journal of Immunology. 163 (12), 6651-6658 (1999).</li><li>Kallen, K. J., Gr&#246;tzinger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+recognition+sites+of+cytokines+are+organized+as+exchangeable+modules.+Transfer+of+the+leukemia+inhibitory+factor+receptor-binding+site+from+ciliary+neurotrophic+factor+to+interleukin-6.">Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6.</a> The Journal of Biological Chemistry. 274 (17), 11859-11867 (1999).</li><li>Gibrat, J. F., Madej, T., Bryant, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surprising+similarities+in+structure+comparison.">Surprising similarities in structure comparison.</a> Current Opinion in Structural Biology. 6 (3), 377-385 (1996).</li><li>Madej, T., Lanczycki, C. 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The generation of chimeric proteins constitutes a versatile technique, which is able to go beyond the limits of truncated proteins to address questions such as the modularity of cytokine-receptor binding domains13. The design of chimeras is a key step in this kind of studies, and requires careful consideration. Preliminary studies to establish functional domains will generally require substitution of broad regions in a first phase, while smaller replacements of variable lengths are more suited to ...

Several types of proteins, including cytokines and growth factors, are grouped in families whose members share similar three-dimensional structures but often exert distinct biological functions1,2. This functional diversity is usually the consequence of small differences in amino acid composition within the molecule&#39;s active sites3. Identification of such sites and functional determinants do not only offer valuable evolutionary insights but also to design more specific agonists and inhibitors4. However, the large number of differences in residue composition frequently found between structurally related proteins complicates this task. Even though constructing large libraries containing hundreds of mutants is nowadays feasible, assessing every single residue variation and combinations of them still remains a challenging and time-consuming effort5.
Techniques assessing the functional importance of large protein regions are thus of value to reduce the number of possible residues to a manageable number6. Truncated proteins have been the most used approach to tackle this issue. Accordingly, regions are considered to be functionally relevant if the protein function under study is affected by the deletion of a particular region7,8,9. However, a major limitation of this method is that deletions can affect the protein&#39;s secondary structure, leading to misfolding, aggregation and the inability to study the intended region. A good example is a truncated version of the cytokine oncostatin M (OSM), in which an internal deletion larger than 7 residues resulted in a misfolded mutant that could not be further studied10.
The generation of chimeric proteins constitutes an alternative and innovative approach that permits the analysis of larger protein regions. The goal of this method is to exchange regions of interest in a protein by structurally related sequences in another protein, in order to assess the contribution of the replaced sections to specific biological functions. Widely used in the field of signaling receptors to identify functional domains11,12, chimeric proteins are particularly useful to study protein families with little amino acid identity but conserved secondary structure. Appropriate examples can be found in the class of interleukin-6 (IL-6) type cytokines, such as interleukin-6 and ciliary neurotrophic factor (6% sequence identity)13 or leukemia inhibitory factor (LIF) and OSM (20% identity)6, on which the following protocol is based.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Labcycler thermocycler</td>
			<td style="width:97px;">Sensoquest</td>
			<td style="width:119px;">011-103</td>
			<td style="width:167px;">Any conventional PCR machine can be employed to carry out this protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 2000c UV-Vis spectrophotometer</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">ND-2000C&#160;</td>
			<td>DNA quantification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GeneRuler 100 bp DNA ladder</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">SM0241</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GeneRuler DNA Ladder Mix</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">SM0331</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AscI restriction enzyme</td>
			<td style="width:97px;">New England Biolabs</td>
			<td style="width:119px;">R0558</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PacI restriction enzyme</td>
			<td style="width:97px;">New England Biolabs</td>
			<td style="width:119px;">R0547</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phusion Hot Start II DNA Polymerase</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">F-549S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP set (100 mM)</td>
			<td style="width:97px;">Invitrogen</td>
			<td>10297018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 DNA ligase</td>
			<td style="width:97px;">Promega</td>
			<td style="width:119px;">M1804</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NucleoSpin Gel and PCR clean-up kit</td>
			<td style="width:97px;">Macherey-Nagel</td>
			<td align="right" style="width:119px;">740609</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">MGC Human LIF Sequence-Verified cDNA (CloneId:7939578), glycerol stock</td>
			<td style="width:97px;">ThermoFisher Scientific</td>
			<td style="width:119px;">MHS6278-202857165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LE agarose</td>
			<td style="width:97px;">Biozym</td>
			<td align="right" style="width:119px;">840004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primers</td>
			<td style="width:97px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td>Custom order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Human Oncostatin M cDNA</td>
			<td style="width:97px;"></td>
			<td style="width:119px;"></td>
			<td>Gift of Dr. Heike Hermanns (Division of Hepatology, University Hospital W&#252;rzburg, Germany)&#160;</td>
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			<td height="42" style="height:42px;width:216px;">pCAGGS vector with PacI and AscI restriction sites</td>
			<td style="width:97px;"></td>
			<td style="width:119px;"></td>
			<td>Gift of Dr. Andr&#233; Schneider (Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany)</td>
		</tr>
	</tbody>
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identification of functional protein regions through chimeric protein construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Construction and generation of a chimeric protein (Figure 1) will be exemplified with two members of the interleukin-6 cytokine family, OSM and LIF, which were the subject of a recently published study6. Figure 2 shows the three-dimensional structure of these proteins. Both molecules adopt the characteristic secondary structure of class I cytokines, with four helices (termed A to D) packed in a bundle and ...

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Identification of Functional Protein Regions Through Chimeric Protein Construction

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

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.

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