June 2009 technical highlight
GFP to the rescue
Modified 'superfolder' green fluorescent protein can quickly establish whether a protein is soluble.
In vitro complementation assay from an in vivo solubility screen of a library. The image of the microplate shows the fluorescence after complementation of the assayed soluble and pellet fraction in vitro. The circled samples in soluble and pellet assay plates correspond to a soluble protein fragment chosen for purification.
Many proteins have multiple domains, but often it is easier to work with a single domain. However, deciding what is and what is not a soluble individual domain is difficult, and bioinformatics programs cannot always provide the answer. Instead, a modified version of GFP can give a quick indication of solubility. This approach is also useful for screening for in vitro protein refolding conditions that reduce misfolding and aggregation.
This system, developed by Los Alamos National Laboratory in collaboration with the PSI, is simple to use and has several advantages. There is no need to subclone the vector for in vivo and then in vitro experiments. The tag does not interfere with the folding of the studied protein or alter its solubility significantly. In addition, the method is quick and easy.
One part of the GFP molecule, a 15 amino-acid GFP fragment termed GFP 11, is cloned into a pTET vector, resulting in an amino-terminal fusion, and is then transformed into Escherichia coli BL21 (DE3) cells that contain the other part of the GFP protein encoded in the pET GFP 1-10 plasmid. This is the 'detector' molecule, which is expressed separately. These fragments associate spontaneously to form a fluorescent signal if the target protein is soluble. The brighter the fluorescence, the more soluble the protein.
Once the two parts bind, the fluorescence is always 'on', which is convenient for downstream applications so there is no need to engineer new GFP fusions. In addition, the sensitivity is such that accurate quantification over a wide range of concentrations is possible. Visible fluorescence appears after only 15 minutes, both in vivo and in vitro.
Recently, the same team from Los Alamos have automated the procedure to reduce the time needed for several of the labor-intensive steps such as in vitro screening of hundreds of clones selected from in vivo screening. They use a robotics system based on the Biomek FX platform and using the SAMI method editor. More information is available from their GFP solubility website.