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Using LANL GFP to Determine Solubility

Overview

LANL's GFP can be used to quickly determine whether an expressed protein is soluble. It does this very simply and elegantly. First, a short section of GFP called a "tether" or "tag" is appended onto the nucleotide sequence of the protein of interest. The tether is about 14 amino acids (a.k.a. S11, strand 11 of GFP) which is too short to effect the dynamics of your expressed protein.

Figure 1: Using split for solubility studies
Figure 1. Split GFP: how a soluble expressed protein will behave in the system.

A vector containing the remaining portion of GFP (a.k.a S1-10, strand 1-10 of GFP) called the "detector" is also inserted into the host cell. Once the target protein and S11, and the remaining portion of GFP, S1-10 are inserted into the host, determining the solubility of the target protein is easy: express your protein of interest (which has the S11 tag) (Fig. 1, #1), followed by inducing the expression of the S1-10 "detector" (Fig. 1, #2).

Split non-soluble
Figure 2. Split GFP: how an insoluble expressed protein will behave in the system.

If your protein is soluble, aggregation will not occur (Fig. 2, #1) and the S1-10 detector will be able to bind to the S11 tag to create a fully functioning, fluorescing GFP (Fig. 1, #3). However, if the expressed protein is not soluble, it will aggregate (Fig. 2, #1), not allowing the S11 tag to interact with the S1-10 detector and no fluorescense will occur (Fig. 2, #3).

Significant efforts have been directed into an automated, robotic-based system to process hundreds of protein samples in a parallel. Using our robotics integration we have developed an automated, high-throughput method for solubility determination. Our robotics system is based on Biomek FX platform, ORCA arm and several peripheral instruments. The Biomek FX methods are integrated with other instruments using SAMI method editor. There are four individual SAMI methods, each with the specific Biomek FX methods. If you are a Biomek FX and/or SAMI user you can download the pdf. file and the scripts here. Any questions on the Biomek FX and SAMI methods and assistance with other robotics platforms should be directed to Dr. Pawel Listwan.

Software Disclaimer:This software has been authored by an employee or employees of Los Alamos National Security, LLC, operator of the Los Alamos National Laboratory (LANL) under Contract No. DE-AC52-06NA25396 with the U.S. Department of Energy.  The U.S. Government has rights to use, reproduce, and distribute this software.  The public may copy, distribute, prepare derivative works and publicly display this software without charge, provided that this Notice and any statement of authorship are reproduced on all copies.  Neither the Government nor LANS makes any warranty, express or implied, or assumes any liability or responsibility for the use of this software.  If software is modified to produce derivative works, such modified software should be clearly marked, so as not to confuse it with the version available from LANL.

Key Characteristics

  • Fast
  • Cheap
  • Reliable
  • Quantifiable
  • Automation and high-throughput sample processing

To learn more, read about published results in our Technical Library.

 

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