Refolding out of guanidine hydrochloride is an effective approach for high‐throughput structural studies of small proteins

Abstract

Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/ refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (Յ18 kD) that are normally soluble in vivo. In addition, refolding the purified proteins by using dialysis against a single buffer allowed us to obtain soluble protein samples of 58% of small proteins that were found in the insoluble fraction in vivo, and 10% of the initial number of proteins provided good heteronuclear single quantum coherence (HSQC) NMR spectra. We conclude that a denaturation/refolding protocol is an efficient way to generate structural samples for high-throughput studies of small proteins.Keywords: Protein structure/folding; protein solubility; protein purification; NMR spectroscopy; circular dichroism spectroscopy; structural proteomics Structure determination using NMR spectroscopy and X-ray crystallography requires the generation of large amounts of soluble recombinant protein. Most often, recombinant proteins for structural biology studies are produced in Escherichia coli because the cells grow rapidly and to high density in inexpensive medium, the expression system is well characterized, and a large number of expression vector systems and mutant host strains are available. In addition, under appropriate conditions, the recombinant protein can comprise >50% of the total cellular protein. However, the use of the E. coli expression system is limited because the target proteins often segregate partially or completely into the insoluble fraction of the cell. Recent studies of protein expression on a large number of prokaryotic and eukaryotic proteins indicate that >50% of recombinant proteins may be found in the insoluble fraction of bacterial cell lysates (Christendat et al. 2000a,b;Yee et al. 2002).Several strategies have been used to increase the probability of producing soluble recombinant proteins in bacterial cells, including co-expressing protein folding modulators, manipulating the temperature of growth and induction, and producing the protein as a fusion with another soluble Reprint requests to: Aled M. Edwards, Banting and Best

A semi-automated method for purification of milligram quantities of proteins on the QIAcube

“…Other general observations of the data reveal that the denaturing method is more effective at purifying AbpSH3-ArkA while the native method appears to be better for AbpSH3. Some of these observations are consistent with other studies that show different proteins have different preferences towards the chosen buffer system [10]. Most striking however is that our new QIAcube method is able to purify equivalent amounts of protein to the corresponding manual based gravity methods and that this method is able to purify orders of magnitude more protein compared to the previous QIAcube spin column method.…”

“…This method can easily be adapted to other types of purifications that use other affinity, hydrophobic or ion exchange resins and should prove to be a versatile method for obtaining sufficient protein for subsequent biochemical analyses. In this study we decided to use this method for both native and denaturing Ni-NTA purifications as different proteins have different preferences for this common affinity resin [10]. …”

Protein folding: Defining a “standard” set of experimental conditions and a preliminary kinetic data set of two‐state proteins

“…The protein was cloned into pet15B (which has a thrombin cleavage site) and expressed and purified as previously described (Maxwell et al 2003). Folding/unfolding rates were determined under the conditions described (Table 1) by laser T‐jump fluorescence spectroscopy (Qiu et al 2002).…”

Biophysical characterization of the isolated C‐terminal region of the transient receptor potential vanilloid 1

“…Several pieces of evidence suggest that this is not the case. Firstly, a similar refolding protocol has been used by others [35,36] and for ourselves (JLN, JB, AC, unpublished results) to refold several proteins (which also accumulate as IBs), and the refolded proteins were biologically active. Second, there are several evidences in the literature where the unfolded predictors predict a folded conformation (Fig.…”