Identification of Functional Protein Regions Through Chimeric Protein Construction

Summary

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.

Abstract

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.

Introduction

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 functions^1,2. 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 f....

Protocol

1. Chimeric Protein Design

1. 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
   1. Access the Protein Data Bank (PDB)¹⁶ European website (https://www.ebi.ac.uk/pdbe/), introduce the name of the protein of interes....

Results

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 .......

Discussion

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 .......

Materials

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)