High-throughput purification of recombinant proteins using self-cleaving intein tags

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.     

Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions

Immobilized metal affinity chromatography (IMAC) of proteins containing poly-histidine fusion tags is an efficient research tool for purifying recombinant proteins from crude cellular feedstocks at laboratory scale. Nevertheless, to achieve successful purification of large amounts of the target protein for critical therapeutic applications that demand the precise removal of fusion tags, it is important to also take into consideration issues such as protein quality, efficiency, cost effectiveness, and optimal affinity tag choice and design. Despite the many considerations described in this article, it is expected that enhanced selectivity, the primary consideration in the field of protein separation, will continue to see the use of IMAC in solving new purification challenges. In addition, the platform nature of this technology makes it an ideal choice in purifying proteins with unknown properties. Finally, the unique interaction between immobilized metal ions and poly-histidine fusion tag has enabled new developments in the areas of biosensor, immunoassay, and other analytical technologies.     

N‐terminal processing of affinity‐tagged recombinant proteins purified by IMAC procedures

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S‐transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine‐containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7‐triazacyclononane (tacn). The use of this tag‐tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli‐expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP‐1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI‐TOF MS analysis of the cleaved products from the DAP‐1 digestion of the recombinant N‐terminally tagged proteins confirmed the complete removal of the tag within 4‐12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn‐based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli‐expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.     

Improving the purification of NAD+-dependent formate dehydrogenase from Candida methylica

The Candida methylica (cm) recombinant wild type formate dehydrogenase (FDH) gene has been cloned into the pQE-2 TAGZyme expression vector and the 6xHis-tagged FDH gene has been overexpressed in JM105 cells to purify the FDH protein more efficiently, by the use of exopeptidases, TAGZyme Purification System, which has allowed the complete removal of the small N-terminal His-tag. After the purification procedure, 1.2 mg/mL cmFDH protein of >95% purity was obtained. The kinetic parameters of cmFDH have been determined by observing the oxidation of the nicotinamide coenzyme at 340 nm. The results have also been compared to the yield of standard vs. affinity purification of FDH.     

The starch-binding domain as a tool for recombinant protein purification

Recombinant protein purification with affinity tags is a widely employed technique. One of the most common tags used for protein purification is the histidine tag (His_tag). In this work, we use a tandem starch-binding domain (SBD_tag) as a tag for protein purification. Four proteins from different sources were fused to the SBD_tag, and the resulting fusion proteins were purified by affinity chromatography using the His_tag or the SBD_tag. The results showed that the SBD_tag is superior to the His_tag for protein purification. The efficient adsorption of the fusion proteins to raw corn starch was also demonstrated, and two fusions were selected to test purification directly using raw starch from rice, corn, potato, and barley. The two fusion proteins were successfully recovered from crude bacterial extract using raw starch, thus demonstrating that the SBD_tag can be used as an efficient affinity tag for recombinant protein purification on an inexpensive matrix.     

Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

Immobilized metal ion affinity chromatography (IMAC) using peptide affinity tags has become a popular tool for protein purification. An important feature dictating the use of a specific affinity tag is whether its structure influences the properties of the target protein to which it is attached. In this work we have studied the influence on protein stability of two novel peptide affinity tags, namely NT1A and HIT2, and compared their effect to the commonly used hexa‐histidine tag, all attached to the C‐terminus of a enhanced green fluorescent protein (eGFP). A comparison of the influence of C‐ or N‐terminal orientation of the tags was also carried out by studying the NT1A tag attached at either terminus of the eGFP. Protein stability was studied utilising guanidine hydrochloride equilibrium unfolding procedures and CD and fluorescence spectroscopy. The novel peptide affinity tags, NT1A and HIT2, and the His₆ tag were found to not affect the stability of eGFP. Although these results are protein specific, they highlight, nevertheless, the need to employ suitable characterisation tools if the impact of a specific peptide tag on the folded status or stability of a recombinant tagged protein, purified by immobilized metal ion affinity chromatographic methods, are to be rigorously evaluated and the appropriate choice of peptide tag made. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Prokaryotic gene fusion expression systems and their use in structural and functional studies of proteins

The ability to express and purify large quantity of proteins in bacteria has greatly impacted many aspects of biological research. These include their use as a source of reagent for biochemical and biophysical studies as well as a source of antigen for antibody production. Currently many different expression systems are available and new ones are being developed. These systems allow inducible expression of a desired protein as a fusion with an affinity tag for simple purification. The affinity tags can generally be removed by specific proteases which recognize cleavage sites engineered between the affinity tag and the desired protein. Presence of tags that encode epitopes of specific antibodies provide additional means for identification of recombinant proteins. This review provides an overview of some of the most commonly utilized expression systems and examples of the use of these proteins in biochemical and biophysical studies. I will also describe other available systems which may provide suitable alternative for expression of recombinant proteins.     

Affinity tag for protein purification and detection based on the disulfide-linked complex of InaD and NorpA

Affinity tags are not only used for the expression and purification of recombinant proteins but also for the detection of protein-protein interactions. Common problems with many affinity tags are excessive length, which may interfere with the structure and function of tagged proteins, and low affinity and/or specificity for primary detection and purification agents. Preliminary results suggest that the C-terminalfive residues of the Drosophila protein NorpA, based on the short, covalent interaction they make with the N-terminal PDZ domain (PDZI) of InaD, are useful as a general affinity tag. First, a PDZI-alkaline phosphatase fusion protein specifically detects both its physiological ligand and a heterologous protein expressing the NorpA C-terminal five residues. The interaction of PDZI with a NorpA-tagged protein is reversible by a reducing agent, which allows nitrocellulose membranes to be stripped completely and reused. In addition, a NorpA-tagged protein can specifically bind to immobilized PDZI resin, while other cellular proteins are washed through. After washing, the NorpA-tagged protein is eluted by a reducing buffer. The NorpA tag's short length makes it the smallest affinity tag available, and its specific and high-affinity interaction with PDZI could yield a powerful system that improves on currently available technology.     

Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures

Many mammalian proteins are multifunctional proteins with biological activities whose characterization often requires in vitro studies. However, these studies depend on generation of sufficient quantities of recombinant protein and many mammalian proteins cannot be easily expressed and purified as full-length products. One example is the Wilm's tumor gene product, WT1, which has proven difficult to express as a full-length purified recombinant protein using standard approaches. To facilitate expression of full-length WT1 we have developed approaches that optimized its expression and purification in Escherichia coli and mammalian cells. First, using a bicistronic vector system, we successfully expressed and purified WT1 containing a C-terminal tandem affinity tag in 293T cells. Second, using a specific strain of E. coli transformed with a modified GST vector, we successfully expressed and purified N-terminal GST tagged and C-terminal 2x FLAG tagged full-length human WT1. The benefits of these approaches include: (1) two-step affinity purification to allow high quality of protein purification, (2) large soluble tags that can be used for a first affinity purification step, but then conveniently removed with the highly site-specific TEV protease, and (3) the use of non-denaturing purification and elution conditions that are predicted to preserve native protein conformation and function.     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.     

Self-cleaving fusion tags for recombinant protein production

Fusion expression is a common practice for recombinant protein production. Some fusion tags confer solubility on the target protein whereas others provide affinity handles that facilitate purification. However, the tag usually needs to be removed from the final product, which involves using expensive proteases or hazardous chemicals and requires additional chromatography steps. Self-cleaving tags are a special group of fusion tags that possess inducible proteolytic activity. Combined with appropriate affinity tags, they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves time, labor and cost. This paper reviews currently available self-cleaving fusion tags for recombinant protein production. For each system, an introduction of its key characteristics and a brief discussion of its advantages and disadvantages is given.     

Design of affinity tags for one-step protein purification from immobilized zinc columns

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.     

Challenges and opportunities in the purification of recombinant tagged proteins

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.     

Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins

The immobilization of a protein by covalent attachment to a support matrix should involve only functional groups of the protein that are not essential for its biological activity. A general strategy for obtaining recombinant proteins designed for oriented covalent grafting onto copolymers was investigated. The rationale involves the definition of seven p24-derived recombinant proteins as fused to either distant or adjacent tags comprising primary amine rich tag consisting of six contiguous lysines suitable for oriented covalent immobilization and a hexa-histidine tag suitable for metal chelate affinity purification. High-level expression, efficient affinity purification, and coupling yields onto maleic anhydride-alt-methyl vinyl ether copolymers higher than 95% were obtained for all proteins. Afterwards, an investigation of the biological features of the immobilized vs. nonimmobilized protein onto the copolymer allowed us to select one bioconjugate which was used in a diagnostic context, i.e., as a capture antigen in an ELISA format test. Sera from 107 HIV-seropositive individuals at various stages of HIV infection, including two seroconversion panels and 104 healthy HIV-seronegative controls, were tested using either RH24 or RK24H-copolymer coated onto the microtiter plate. These assays showed that the use of such a protein-copolymer bioconjugate allowed detection of lower antibody titers than the RH24 protein, illustrating the potential of applications of such doubly tagged proteins. Thus, a set of expression vectors was designed containing four different combinations of hexa-lysine and hexa-histidine tags and a multiple cloning site, allowing the production of different recombinant fusion proteins suitable for biological reactivity conservation after immobilization.     

Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein production and purification

Although most commonly used for protein production, expression of soluble and functional recombinant protein in Escherichia coli is still a major challenge. The development and application of fusion tags that can facilitate protein expression and solubility partly solve this problem, however, under most circumstance, the fusion tags have to be removed by proteases in order to use the proteins. Because the tag removal using proteases increases cost and introduces extra purification steps, it remains a significant problem that must be resolved before being widely used in industry production. Ubiquitin and SUMO have been successfully used to enhance protein expression and solubility. In the last decades, intein has also been widely used in protein production for its self-cleavage property, which could help to remove the fusion tag without any protease. Here, we take the advantages of ubiquitin, SUMO2 and intein in protein expression. We constructed tandem ubiquitin-intein and SUMO2-intein fusion tags, and chose human MMP13 (amino acid 104-274) and eGFP as the passenger proteins that fused to the C-terminus of the tags. These constructs were expressed in E. coli and both MMP13 and eGFP expression and solubility were evaluated. Both tags showed the ability to enhance the solubility of MMP13 and eGFP and improve the expression of eGFP, and the SUMO2-intein having a more significant effect. Both ubiquitin-intein-eGFP and SUMO2-intein-eGFP were purified using Ni-NTA column chromatography and self-cleavaged by changing pH. The recombinant un-tagged eGFP were released and eluted with high homogeneity. In summary, ubiquitin-intein and SUMO2-intein are convenient and useful fusion tags that can enhance the expression, solubility and improve the purification process of the model heterologous protein and these tags may have a good prospect in protein production.     

Inhibition of tobacco etch virus protease activity by detergents

Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.     

Lectin-based affinity tag for one-step protein purification

The production of pure protein is indispensable for many applications in life sciences, however protein purification protocols are difficult to establish, and the experimental procedures are usually tedious and time-consuming. Therefore, a number of tags were developed to which proteins of interest can be fused and subsequently purified by affinity chromatography. We report here on a novel lectin-based affinity tag using the D-mannose-specific lectin LecB from Pseudomonas aeruginosa. A fusion protein was constructed consisting of yellow fluorescent protein and LecB separated by an enterokinase cleavage site. This protein was overexpressed in Escherichia coli Tuner (DE3), and the cell extract was loaded onto a column containing a mannose agarose matrix. Electrophoretically pure fusion protein at a yield of 24 mg/L culture was eluted with a D-mannose containing buffer The determination of equilibrium adsorption isotherms revealed an association constant of the lectin to the mannose agarose matrix of Ka = 3.26 x 10(5)/M. Enterokinase treatment of the purified fusion protein resulted in the complete removal of the LecB-tag. In conclusion, our results indicate that the lectin LecB of P. aeruginosa can be used as a tag for the high-yield one-step purification of recombinant proteins.     

High level expression and single-step purification of hexahistidine-tagged L-2-hydroxyisocaproate dehydrogenase making use of a versatile expression vector set

Affinity tags as fusions to the N- or C-terminal part of proteins are valuable tools to facilitate the production and purification of proteins. In many cases, there may be the necessity to remove the tag after protein preparation to regain activity. Removal of the tag is accomplished by insertion of a unique amino acid sequence that is recognized and cleaved by a site specific protease. Here, we report the construction of an expression vector set that combines N- or C-terminal fusion to either a hexahistidine tag or Streptag with the possibility of tag removal by factor Xa or recombinant tobacco etch virus protease (rTEV), respectively. The vector set offers the option to produce different variants of the protein of interest by cloning the corresponding gene into four different Escherichia coli expression vectors. Either immobilized metal affinity chromatography or streptactin affinity chromatography can be used for the one-step purification. Furthermore, we show the successful application of the expression vector for C-terminal hexahistidine tagging. The expression and purification of His-tagged L-2-hydroxyisocaproate dehydrogenase yields fully active enzyme. The tag removal is here accomplished by a derivative of rTEV.     

Removal of cleavage slow points from affinity tags used in the IMAC purification of recombinant proteins

The complete enzymatic removal of affinity tags from tagged recombinant proteins is often required but can be challenging when slow points for cleavage exist. This study documents a general approach to remove N‐terminal tags from recombinant proteins specifically designed to be efficiently captured by IMAC resins. In particular, site‐directed mutagenesis procedures have been used to modify the amino acid sequence of metal binding tags useful in IMAC purifications of recombinant proteins with the objective to increase cleavage efficiency with the exopeptidase, dipeptidyl aminopeptidase 1. These tags were specifically developed for application with borderline metal ions, such as Ni²⁺ or Cu²⁺ ions, chelated to the immobilized ligands, 1,4,7‐triazacyclononane (tacn) and its analogs. Due to the ability to control cleavage site structure and accessibility via site directed mutagenesis methods, these procedures offer considerable scope to obtain recombinant proteins with authentic native N‐termini, thus avoiding any impact on structural stability, humoral and cellular immune responses, or other biological functions. Collectively, these IMAC‐based methods provide a practical alternative to other procedures for the purification of recombinant proteins with tag removal. Overall, this approach is essentially operating as an integrated down‐stream purification capability.