Acquisition of Multidimensional NMR Data on GST-Fused Proteins

Abstract

With the advances in biological sciences, there is a prevalent demand for large quantities of biologically active polypeptides. High throughput screenings in fields such as proteomics, genomics and bioinformatics give a large variety of proteins that could potentially have therapeutic, diagnostic or industrial purposes and recombinant proteins have become the preferred way of production.Glutathione S-Transferase (GST) is not only a very commonly used affinity tag to increase expression yields, but is also known to enhance the solubility of the protein of interest making it a valuable tool in the pursuit of purifying recombinant proteins.Multidimensional NMR spectroscopy is a popular technique to elucidate the 3D structure of proteins in solution. However, obtaining the structural information of peptides and small proteins can be challenging. We have developed a new method, which facilitates the acquisition of NMR based structural information on protein(s) of interest by using the GST-tagged target protein. Our results show that the carrier protein GST surprisingly does not seem to have an effect on the quality of NMR data of its associated fusion partner. It is well known that GST isozymes exist as dimers, but there has been little research on the state of GST-fused proteins. Our results suggest that the GST-tag and the fused partner can be observed as two entities in multidimensional NMR spectra. Furthermore, small angle X-ray scattering (SAXS) supports that GST-fused proteins are predominantly dimers in solution. We predict the loss of signals in the 1H-15N HSQC spectrum corresponding to the GST carrier is primarily due to decrease in the T2 relaxation rate.