Translational fusions between a protein of interest and a reporter protein are used to determine the subcellular location of proteins in vivo. However, fusion to a reporter protein sometimes renders the protein of interest nonfunctional because the addition of the reporter protein interferes with proper protein folding, enzymatic activity, or protein–protein interactions. You have constructed a fusion between your protein of interest and a reporter gene. How will you show that the fusion protein retains its normal biological function?

Key Concepts

Protein Functionality

Protein functionality refers to the ability of a protein to perform its biological role, which can include enzymatic activity, structural support, or interaction with other molecules. Assessing functionality is crucial when studying fusion proteins, as the addition of a reporter can disrupt these roles. Techniques such as enzyme assays or interaction studies can help determine if the fusion protein retains its original function.

Protein Folding

Protein folding is the process by which a polypeptide chain acquires its functional three-dimensional structure. Proper folding is essential for protein activity, as misfolded proteins can lead to loss of function or aggregation. When creating fusion proteins, it is important to ensure that the reporter does not hinder the folding process, which can be evaluated through methods like circular dichroism or fluorescence spectroscopy.

Subcellular Localization

Subcellular localization refers to the specific location within a cell where a protein is active or functions. Understanding where a protein localizes is vital for elucidating its role in cellular processes. Techniques such as fluorescence microscopy can be employed to visualize the localization of the fusion protein, helping to confirm that it is correctly positioned within the cell and retains its biological function.