One-Step Tag-Free Protein PurificationsThe need to produce recombinant proteins that are good drug candidates or targets has dramatically increased in the post-genome era.by Xuemei He, Lei Li, Lily Woo, William Strong, Bio-Rad Laboratories, Inc., and Natalia Oganesyan, Potomac Affinity Proteins Kevin Ridge at the Univ. of Texas Health Science Center in Houston studies the structure and function of the visual photoreceptor rhodopsin as a model for understanding G-protein coupled receptors (GPCRs). While Ridge’s interest may be more academic, pharmaceutical companies are also interested in GPCRs such as rhodopsin as they expect GPCRs to average about 40% of their drug discovery efforts.
Once a GPCR is activated by an external signal, it changes conformation and binds to a heterotrimeric G protein that transmits the signal inside the cell. To shut off the signal, arrestin binds to a phosphorylated form of the GPCR. Ridge’s goal is to produce high-resolution structures of the activated conformations of rhodopsin, heterotrimeric G proteins and arrestin. NMR elucidation of these structures first requires purification of overexpressed, recombinant proteins.
Figure 1. Encoding sequence of target protein is cloned into Profinity eXact pPAL7 expression vector.
Click to enlarge. | Traditionally, to simplify recombinant protein purification, several genetically engineered affinity tags, or purification tags, are used. Commonly used tags are polyhistidine (His), glutathione-S-transferase (GST), and the FLAG antibody peptide epitope. Researchers have widely adopted the inclusion of affinity tags for their ability to provide a generic (vs. customized) approach to purification, ease of use, high specificity and low economic cost.
Issues with tags
The tag is fused to the N- or C-terminus of the protein of interest, allowing the fusion protein to be purified to near homogeneity in a single-step procedure using a resin with strong binding avidity and selectivity to the tag. While gene fusion technology has contributed towards the elucidation of protein structure and function and potential drug candidates, challenges still exist. The inconsistencies related to cleavage of the fusion partner (tag) and its efficient removal and separation from the protein of interest remain as major disadvantages of working with protein fusions.
The tag may alter protein conformation, affect biologically important functions, or interfere with protein crystallization.
Figure 2. Chromatograms from the purification of GFP mutant protein from E.coli (left) and SDS-PAGE analysis of the fractions from the purification of GFP (right).
Click to enlarge. | The most popular method to remove the tag involves building a protease cleavage site between the tag and the target protein within the expression vector, and cleaving the resultant fusion protein using purified preparations of the cognate protease specific to the engineered site. The most frequently used processing proteases for this purpose are the tobacco etch virus protease, thrombin, factor Xa and enterokinase.
Although these tag-removal systems alleviate problems associated with the presence of the tag in the final purified protein, they have several principal drawbacks: 1) reduced stability of the target protein; 2) extended length of purification protocols due to additional cleavage and protease-removal steps, which may hamper high-throughput purification approaches and result in loss of target protein; and 3) the nature of protease cleavage mechanisms often result in generation of protein products that contain extra residues on their N-termini.
In Ridge’s case, he first attempted purification of overexpressed rhodopsin system proteins, including the alpha subunit of the heterotrimeric G protein, using a His-tag system. After concentrating it to several milligrams per milliliters for NMR, he encountered a problem commonly observed by structural biologists: The proteins aggregated and unfolded. Rather than use cleavage enzymes to remove the His-tag, he tried a new fusion tag from Bio-Rad called the Profinity eXact fusion-tag purification system, which in one step produces protein without any tag. For Ridge, the result was more-soluble, functional protein in a shorter period of time.
Affinity purification and tag removal
The Profinity eXact purification resin is an integral part of the Profinity eXact fusion-tag system for the expression and purification of recombinant proteins overproduced in Escherichia coli cells. It essentially incorporates affinity purification and tag removal in a single step to alleviate bottlenecks in the generation of native recombinant proteins from parent fusion proteins following affinity chromatography purification.
The encoding sequence of a target protein is cloned into the Profinity eXact pPAL7 expression vector downstream from the Profinity eXact tag, resulting in an N-terminally tagged fusion protein. The mutant serine protease immobilized onto Profinity eXact purification resin selectively interacts with the cognate Profinity eXact tag (KD <100 pM). Once contaminants from the expression host cells are removed by a post-binding wash, proteolysis is triggered precisely at the junction between the affinity tag and target protein by the addition of fluoride or azide anion. With the 8kD Profinity eXact tag retained by the immobilized protease, only the desired native recombinant protein is eluted from the column and is available for downstream applications, often without further manipulation.
Purification under native conditions
A variety of gene sequences from both prokaryotic and eukaryotic species for the expression and purification of their protein products have been cloned using the Profinity eXact fusion-tag system. These proteins also differed in terms of their polypeptide molecular mass and oligomeric state. Some of these proteins have been well characterized, while the structural properties and biological functions of others have yet to be determined or confirmed.
The tag-free recombinant proteins were obtained at satisfactory purity using a generic protocol outlined in the Profinity eXact fusion-tag system manual, with no substantial optimization performed for each individual protein. For very large (>100 kD) proteins, such as the tetrameric beta-galactosidase, binding time was extended to improve target protein yield. Based on the wide variety of proteins purified using the Profinity eXact system, the binding of fusion proteins to the resin using the Profinity eXact tag appeared to be very selective, independent of fusion protein sequence or structure.
Contaminants from expression host cells were easily removed by washing the loaded resin with Profinity eXact bind/wash buffer.
Table 1.Recombinant proteins and putative functional domains purified using Profinity eXact mini spin columns.
Click to enlarge.
| The on-column cleavage of bound fusion proteins was found to be efficient and consistent, even though the primary sequences and tertiary structures of proteins are dramatically different. Unlike the reported promiscuous cleavage behavior exhibited by some commercially available endoproteases, such as recombinant enterokinase and factor Xa, the Profinity eXact tag precludes nonspecific cleavage of the desired proteins because of the extensive additional interactions between other Profinity eXact tag amino acids outside of the core cleavage recognition motif and the subtilisin protease. In fact, the data provide evidence supporting this notion. The purified polyADP-ribose polymerase mutant and apiose synthase exhibit their expected biological activities, which indicate that the physical and biological properties of these proteins have been maintained under the mild chromatographic condition in this one-step purification and tag-removal process.
The purification of a tag-free GFP mutant with a 1-mL Bio-Scale Mini Profinity eXact cartridge on a Biologic DuoFlow system resulted in about 4 mg of protein with purity of 98%. Moreover, other than total yield, virtually no difference in performance between cartridge and mini spin column formats was found, as is evident in the preparation of native MBP; This is an important consideration when progressing from small-scale to more preparative-scale purifications.
Understanding the 3-D structures of protein targets plays a major role in drug discovery and design. Many pharmaceutical companies employ structure-based approaches to assist in developing good drug candidates. The need, therefore, to produce recombinant proteins known to be good drug candidates or targets has dramatically increased in the post-genome era. For more information visit www.bio-rad.com
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