Troubleshooting Tip 4: Fluorescently-tagged Protein Forms Aggregates

Problem

These micrographs show living tissue labeled with a plasma membrane targeting signal fused to the fluorescent protein mEosFP. mEosFP misfolds at high temperatures and forms aggregates (red arrowheads, right panel), whereas temperatures below 30ºC (left panel) minimize this effect. Here we discuss common issues that one needs to consider in order to minimize the aggregation of fluorescent protein (FP) fusion products.

Possible Causes and Solutions

I. Oligomerization of the FP moiety

Does the FP I chose oligomerize? Most FPs that have been discovered to date have a tendency to oligomerize. Once expressed, the formation of dimers or higher-order oligomers by the FP moiety of the fusion protein can lead to the formation of aggregates. One straightforward way to enforce FP monomerization is to introduce mutations that reduce or eliminate self-association (e.g. the A206K mutation in the jellyfish-derived GFP variants (Zacharias et al., 2002)). In the case of FPs that exist naturally as obligate dimers or tetramers (e.g. most reef coral and anemone variants) one can generate vectors containing two sequential coding regions for the FP separated by a short sequence of non-specific amino acids (tandem dimer). Upon expression, the fused FPs preferentially bind to each other to form an intramolecular dimer that performs as a monomer, although at twice the size. Such an approach has been succesfully used for various FPs (e.g. tdTomato, tdEos).

II. Oligomerization of the fusion partner

Does my protein of interest oligomerize? If the fusion partner itself also tends to oligomerize or participates in natural oligomer formation (such as histones or tubulin), there is increased risk that fusion proteins are cross-linked to form massive aggregates. Use strictly monomeric versions of FPs and further test different FPs to identify the ones where aggregation is absent.

III. Total expression levels

Do the expression levels of my FP fusion match the endogenous expression levels of the protein of interest? Transient overexpression of FP fusions typically results in higher protein levels than the endogenous ones. Such high levels saturate the cell's folding machinery and can lead to protein aggregation. It is best to control the expression levels of the fusion protein depending on the model system used (establishment of cell lines from low-level expressing clones, use of native promoters, use of lower temperature if possible).

IV. Local protein concentration

Where is my protein localized? Fusion proteins with FPs that weakly dimerize (e.g. GFP variants) may not aggregate as long as the local concentration is low. However, when such FPs are targeted to specific cellular compartments, such as the plasma membrane or organelle membranes, the local, effective concentration of the protein can be high enough to induce dimerization and aggregation. Use monomeric versions of FPs and further test various FPs to identify the ones where aggregation is absent/minimized.

V. Temperature

Is the temperature optimized for the FP I chose? There are temperature-sensitive FPs such as mEosFP which works below 30ºC. Work at lower temperature if possible, or use alternative variants.

VI. Site of fusion

Does it matter where I fuse the FP? Tagging the N-terminus versus the C-terminus, or a cytosolic versus an extracellular or luminal domain can compromise protein structure and thus lead to aggregation. Control experiments should determine which site is best tolerated.

Finally, one needs to carefully consider whether an aggregate-like distribution of a fusion protein is indeed an artifact (due to any of the abovementioned reasons) or if it reflects a distinct functional organization of the protein of interest (assuming its fusion to a strictly monomeric FP).

More information on fluorescent proteins and their properties can be found in Current Protocols in Cell Biology, Unit 21.5. Information on how to design fluorescent protein fusions can be found in Current Protocols in Cell Biology, Unit 21.4.

Visit www.currentprotocols.com for tools, calculators, apps, videos, and information on all Current Protocols methods.

Contributed by: Manos Mavrakis