Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage

While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale‐up have promoted the development of alternative non‐chromatographic separation methods. Here we developed a new non‐chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo‐responsive elastin‐like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin–dockerin pairs, which are high‐affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin‐tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self‐cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used. Biotechnol. Bioeng. 2012; 109: 2829–2835. © 2012 Wiley Periodicals, Inc.     

Characterization of a dockerin-based affinity tag: application for purification of a broad variety of target proteins

Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca(2+)-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K(A)) of 7.7 × 10(8)M(-1), calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum β-glucosidase, purified using this approach, was tested and found to be similar to that of a β-glucosidase preparation (without the ΔDoc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca(2+)-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification.     

A pH‐induced,intein‐mediated expression and purification of recombinant human epidermal growth factor in Escherichia coli

Human epidermal growth factor (hEGF) is a cellular factor that promotes cell proliferation and has been widely used for the treatment of wounds, corneal injuries, and gastric ulcers. Recombinant hEGF (rhEGF) has previously been expressed using the pTWIN1 system with pH‐induced intein and a chitin‐binding domain. The rhEGF protein can be purified by chitin affinity chromatography because of the high affinity between the chitin‐binding domain fusion‐tag and the column. However, uncontrolled cleavage presents a major problem with this method. To overcome this problem, a novel purification method has been developed for a pH‐induced intein tag rhEGF that is expressed in Escherichia coli. Following purification by denaturation of inclusion bodies, the fusion protein is renatured and simultaneously induced to self‐cleave by dialysis. Further purification of rhEGF is achieved by heat treatment and ion‐exchange chromatography. Our results show that the purity of rhEGF obtained through this method is over 98% and the quantity of purified rhEGF is 248 mg from a 1 L culture or 2,967 mg from a 12 L culture. Therefore, we conclude that we have developed an efficient purification method of rhEGF, which may be used for the purification of other heat‐resistant and acid‐resistant recombinant proteins. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:758–764, 2015     

Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag

A self‐cleaving elastin‐like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP α subunit was tagged with a self‐cleaving ELP‐intein tag and coexpressed with the β, β′, and ω subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP‐intein purification. Thus, this simple ELP‐based method should be applicable for the nonchromatographic purification of other recombinant, in vivo‐assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large‐scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.     

Building a foundation for structure‐based cellulosome design for cellulosic ethanol: Insight into cohesin‐dockerin complexation from computer simulation

The organization and assembly of the cellulosome, an extracellular multienzyme complex produced by anaerobic bacteria, is mediated by the high‐affinity interaction of cohesin domains from scaffolding proteins with dockerins of cellulosomal enzymes. We have performed molecular dynamics simulations and free energy calculations on both the wild type (WT) and D39N mutant of the C. thermocellum Type I cohesin‐dockerin complex in aqueous solution. The D39N mutation has been experimentally demonstrated to disrupt cohesin‐dockerin binding. The present MD simulations indicate that the substitution triggers significant protein flexibility and causes a major change of the hydrogen‐bonding network in the recognition strips—the conserved loop regions previously proposed to be involved in binding—through electrostatic and salt‐bridge interactions between β‐strands 3 and 5 of the cohesin and α‐helix 3 of the dockerin. The mutation‐induced subtle disturbance in the local hydrogen‐bond network is accompanied by conformational rearrangements of the protein side chains and bound water molecules. Additional free energy perturbation calculations of the D39N mutation provide differences in the cohesin‐dockerin binding energy, thus offering a direct, quantitative comparison with experiments. The underlying molecular mechanism of cohesin‐dockerin complexation is further investigated through the free energy profile, that is, potential of mean force (PMF) calculations of WT cohesin‐dockerin complex. The PMF shows a high‐free energy barrier against the dissociation and reveals a stepwise pattern involving both the central β‐sheet interface and its adjacent solvent‐exposed loop/turn regions clustered at both ends of the β‐barrel structure.     

Engineered proteins containing the cohesin and dockerin domains from Clostridium thermocellum provides a reversible, high affinity interaction for biotechnology applications

The cohesin-dockerin interaction, which is responsible for the formation of the cellulosome complex of cellulolytic bacteria, is a calcium-dependent, high affinity interaction. In this study, the cohesin (Cip7) and dockerin (Doc) domains of Clostridium thermocellum were fused to the cellulose-binding domain (CBD) of C. cellulovorans and the antibody-binding domain, protein LG, respectively, to form CBD-Cip7 and LG-Doc. Immobilised CBD-Cip7 was able to bind LG-Doc and subsequently antibody as determined using surface plasmon resonance. Binding was reversed by the removal of Ca2+ with EDTA. The dockerin containing fusion protein was affinity purified using an immobilised cohesin domain. Elution of the LG-Doc from the cohesin column was with EDTA. This affinity chromatography was repeated using an LG-dockerin column for the purification of cohesin fusion protein. The fusion proteins created in this report have shown that the properties of the cohesin and dockerin domains can be transferred to other protein domains and that the interaction between the cohesin and dockerin is specific, Ca2+ -dependent and reversible. We have shown that the cohesin-dockerin interaction has several properties making it suitable for use in recombinant fusion protein production and purification.     

Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

利用小肽控制的基于蛋白质内含子非层析标签制备重组蛋白(英文)

基于蛋白质内含子的蛋白质纯化自我断裂标签已经被广泛使用超过15年之久.但这一系统体内表达过程的提前断裂一直是限制这一技术广泛应用的瓶颈,特别是在需要高温表达和长表达周期的真核表达系统中.本研究介绍了一种利用小肽控制的基于蛋白质内含子和非层析标签ELP(elastin-like polypeptide)的自我断裂系统.在这一系统中,蛋白质内含子的体内外活性严格受到其结构互补小肽控制.在体内表达不含有互补小肽时,蛋白质内含子不具有活性;而在体外添加结构互补小肽,蛋白质内含子结构恢复并发生C端断裂反应释放目的蛋白.由于非层析标签ELP的引入,因此整个纯化过程可以简单地通过几步机械沉淀完成.此外,这一系统反应pH、小肽与前体蛋白之间的摩尔比及断裂速率也一并进行了系统的研究.     

A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification

Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein–ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.     

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.     

Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step.

A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.     

Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags

We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.     

In‐solution antibody harvesting with a plant‐produced hydrophobin–Protein A fusion

Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bifunctional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low‐cost and scalable antibody capturing in solutions. Immunoglobulin‐binding Protein A is widely used in affinity‐based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two‐phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana. The hydrophobins enhanced accumulation up to 35‐fold, yielding up to 25% of total soluble protein. Both fused and nonfused Protein A accumulated in protein bodies. Hence, the increased yield could not be attributed to HFB‐induced protein body formation. We also demonstrated production of HFBI–Protein A fusion protein in tobacco BY‐2 suspension cells in 30 l scale, with a yield of 35 mg/l. Efficient partitioning to the surfactant phase confirmed that the fusion proteins retained the amphipathic properties of the hydrophobin block. The reversible antibody‐binding capacity of the Protein A block was similar to the nonfused Protein A. The best‐performing fusion protein was tested in capturing antibodies from hybridoma culture supernatant with two‐phase separation. The fusion protein was able to carry target antibodies to the surfactant phase and subsequently release them back to the aqueous phase after a change in pH. This report demonstrates the potential of hydrophobin fusion proteins for novel applications, such as harvesting antibodies in solutions.     

Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction

Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module.     

Cellulosome from Clostridium cellulolyticum: molecular study of the Dockerin/Cohesin interaction.

Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent. In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function. This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction. Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin. These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other. Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment. Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.     

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self‐assembling peptides and a C‐terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self‐assembling peptide. Upon simple separation, the target protein or peptide with an authentic N‐terminus is then released in the solution by intein‐mediated cleavage. Different combinations of four self‐assembling peptides (ELK16, L₆KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI‐CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI‐CM, which has pH‐shift inducible cleavage, was found to work well with three self‐assembling peptides (L₆KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx‐strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N‐terminus, which is especially effective for peptides of 30‐100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.     

Protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin

Elastin-like polypeptides (ELPs) undergo a reversible, soluble-to-insoluble phase transition in aqueous solution upon heating through a characteristic transition temperature (T(t)). Incorporating a terminal ELP expression tag into the gene of a protein of interest allows ELP fusion proteins to be purified from cell lysate by cycles of environmentally triggered aggregation, separation from solution by centrifugation, and resolubilization in buffer. In this study, we examine the effect of ELP length on the expression and purification of a thioredoxin-ELP fusion protein and show that reducing the size of the ELP tag from 36 to 9 kDa increases the expression yield of thioredoxin by 4-fold, to a level comparable to that of free thioredoxin expressed without an ELP tag, while still allowing efficient purification. However, truncation of the ELP tag also results in a more complex transition behavior than is observed with larger tags. For both the 36 kDa and the 9 kDa ELP tag fused to thioredoxin, dynamic light scattering showed that large aggregates with hydrodynamic radii of approximately 2 microm form as the temperature is raised to above the T(t). These aggregates persist at all temperatures above the T(t) for the thioredoxin fusion with the 36 kDa ELP tag. With the 9 kDa tag, however, smaller particles with hydrodynamic radii of approximately 12 nm begin to form at the expense of the larger, micron-size aggregates as the temperature is further raised above the T(t). Because only large aggregates can be effectively retrieved by centrifugation, efficient purification of fusion proteins with short ELP tags requires selection of solution conditions that favor the formation of the micron-size aggregates. Despite this additional complexity, our results show that the ELP tag can be successfully truncated to enhance the yield of a target protein without compromising its purification.     

Production of functional agarolytic nano-complex for the synergistic hydrolysis of marine biomass and its potential application in carbohydrate-binding module-utilizing one-step purification

Marine algal polysaccharides are promising alternative resources to terrestrial biomass. Agarolytic enzymes degrade agarose to various kinds of oligosaccharides. In this study, we expressed miniCbpA, a recombinant scaffolding protein from Clostridium cellulovorans, in Escherichia coli. To assemble the agarolytic complex via cohesin–dockerin interaction, we constructed a chimeric agarase cAgaB from Zobellia galactanivorans containing the catalytic domain of AgaB fused with a dockerin domain from C. cellulovorans EngB. The assembly of functional agarolytic complexes increased the activity against the agar substrate approximately 1.4-fold compared with that for the corresponding enzymes alone. The carbohydrate-binding module (CBM) of miniCbpA was used as a tag for CBM-utilizing one-step purification using cellulose as a support. This is the first report on the formation of agarolytic complexes using the cohesin–dockerin interaction system. The assembly of agar-degrading complexes will lead to the commercial production of useful products from agar biomass at low costs.     

Recombinant protein purification by self-cleaving aggregation tag

A simple technique is presented for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. This method is based on a reversibly precipitating, self-cleaving purification tag. The tag is made up of two components: an elastin-like polypeptide (ELP), which reversibly self-associates in high-salt buffers at temperatures above 30 degrees C; and an intein, which causes the ELP tag to self-cleave in response to a mild pH shift. Thus, a tripartite ELP-intein-target protein precursor can be purified by cycles of salt addition, heating and centrifugation. Once purified, intein-mediated self-cleavage, followed by precipitation of the cleaved ELP tag, allows easy and effective isolation of the pure, native target protein without the need for chromatographic separations. Recoveries of 50-100 mg of cleaved, native target protein per liter of shake-flask culture have been achieved for over a dozen proteins, typically in 8-24 h depending on specific process parameters.