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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 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.
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'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'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.
1. Chimeric Protein Design
2. Preparation for Molecular Cloning
3. Polymerase Chain Reaction (PCR) Amplification of the Individual DNA Fragments Forming the Chimera
4. PCR Amplification to Generate the Chimeric DNA Sequence
5. Insertion of the Chimeric DNA into an Expression Vector
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 ...
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 ...
The authors have nothing to disclose.
This work was supported by the Max Planck Society and the Schüchtermann-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., Lörchner, H., Braun, T. & Pöling, 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. © the Authors.
Name | Company | Catalog Number | Comments |
Labcycler thermocycler | Sensoquest | 011-103 | Any conventional PCR machine can be employed to carry out this protocol |
NanoDrop 2000c UV-Vis spectrophotometer | ThermoFisher Scientific | ND-2000C | DNA quantification |
GeneRuler 100 bp DNA ladder | ThermoFisher Scientific | SM0241 | |
GeneRuler DNA Ladder Mix | ThermoFisher Scientific | SM0331 | |
AscI restriction enzyme | New England Biolabs | R0558 | |
PacI restriction enzyme | New England Biolabs | R0547 | |
Phusion Hot Start II DNA Polymerase | ThermoFisher Scientific | F-549S | |
dNTP set (100 mM) | Invitrogen | 10297018 | |
T4 DNA ligase | Promega | M1804 | |
NucleoSpin Gel and PCR clean-up kit | Macherey-Nagel | 740609 | |
MGC Human LIF Sequence-Verified cDNA (CloneId:7939578), glycerol stock | ThermoFisher Scientific | MHS6278-202857165 | |
LE agarose | Biozym | 840004 | |
Primers | Sigma-Aldrich | Custom order | |
Human Oncostatin M cDNA | Gift of Dr. Heike Hermanns (Division of Hepatology, University Hospital Würzburg, Germany) | ||
pCAGGS vector with PacI and AscI restriction sites | Gift of Dr. André Schneider (Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany) |
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