Characterization of polycationic amino acids fusion systems for ion-exchange purification of cyclodextrin glycosyltransferase from recombinant Escherichia coli

Fusion proteins with charged polycationic amino acid tails were constructed for the purpose of simple ion-exchange purification with high purity. A number of positively charged lysine and arginine tails were fused to the C-terminus of cyclodextrin glycosyltransferase (CGTase) derived from Bacillus macerans and expressed in Escherichia coli. The ionic binding forces provided by the tails allowed the selective recovery of CGTase from recombinant E. coli cell extracts, while CGTase by itself could not bind to the cation exchanger at neutral pH. The type of amino acids used and the length of the tail directly affected the purification factors. Most intracellular proteins of E. coli adsorbed on the cation exchanger could be removed by washing with 400 mM NaCl solution at pH 7.4, suggesting that a fusion partner suitable for purification purpose should be provided with high binding strength and the maintenance of adsorption by washing with NaCl solution. Among the fusion CGTases constructed, the CGTK10ase containing 10 lysine residues provided sufficiently high binding strength to allow purification to its homogeneity through simple ion-exchange chromatography.     

Production of cyclodextrin by poly-lysine fused Bacillus macerans cyclodextrin glycosyltransferase immobilized on cation exchanger

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the V_m of the immobilized CGTase by 40% without a change in K_m. The activation energies of thermal deactivation (E_a) were 41.4, 28.1, and 25.9 kcal mol⁻¹, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l⁻¹ h⁻¹ was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min⁻¹ (D = 240 h⁻¹) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0.     

Electrostatic interaction-induced inclusion body formation of glucagon-like peptide-1 fused with ubiquitin and cationic tag

In an attempt to produce glucagon-like peptide-1 (GLP-1) using recombinant Escherichia coli, ubiquitin (Ub) as a fusion partner was fused to GLP-1 with the 6-lysine tag (K6) for simple purification. Despite the high solubility of ubiquitin, the fusion protein K6UbGLP-1 was expressed mainly as insoluble inclusion bodies in E. coli. In order to elucidate this phenomenon, various N- and C-terminal truncates and GLP-1 mutants of K6UbGLP-1 were constructed and analyzed for their characteristics by various biochemical and biophysical methods. The experiment results obtained in this study clearly demonstrated that the insoluble aggregation of K6UbGLP-1 was attributed to the electrostatic interaction between the N-terminal 6-lysine tag and the C-terminal GLP-1 before the completion of folding which might be one of the reasons for protein misfolding frequently observed in many foreign proteins introduced with charged amino acid residues such as the His tag and the protease recognition sites. The application of a cation exchanger for neutralizing the positive charge of the 6-lysine tag in solid-phase refolding of K6UbGLP-1 successfully suppressed the electrostatic interaction-driven aggregation even at a high protein concentration, resulting in properly folded K6UbGLP-1 for GLP-1 production.     

Production of cyclodextrins in ultrafiltration membrane reactor containing cyclodextrin glycosyltransferase from Bacillus macerans

An enzyme reactor installed with ultrafiltration membrane was developed to produce alpha-, beta-, and gamma-cyclodextrins (CDs) from soluble starch by Bacillus macerans cyclodextrin glycosyltransferase (CGTase) tagged with 10 lysines at its C-terminus (CGTK10ase). Ultrafiltration membrane YM10 with 10,000 of molecular cutoff was chosen for membrane modification and CD production. A repeated-batch type of the enzyme reaction with free CGTK10ase resulted in a alpha-CD yield of 24.0 (+/-1.5)% and a productivity of 4.68 (+/-0.88) g/l-h, which were 7 times higher that those for CGTK10ase immobilized on modified YM10 membrane. Addition of 1- nonanol increased CD yields by 30% relative to the control, which might be due to prevention of the reversible hydrolysis of CDs.     

Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag

A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.     

High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.     

Solid-phase refolding of poly-lysine tagged fusion protein of hEGF and angiogenin

A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinantE. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solidphase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below 30%, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to theN-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5–3 times to about 88%. Besides the intrinsic affinity of angiogenin to heparin the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, fromc.a 4.8% in the conventional refolding of the untagged fusion protein to 63.6%. The process was highly reproducible. The refolded protein in the column eluate retained R Nase bioactivity, of angiogenin.     

Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.     

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.     

Protein Production in Yarrowia lipolytica Via Fusion to the Secreted Lipase Lip2p

We established a strategy for protein production and purification via expression in Yarrowia lipolytica as Lip2p fusion protein. To evaluate the expression system a cysteine-rich miniprotein, an antibody fragment and an enzyme showing galactose oxidase activity were chosen. These proteins have varying disulfide bond content, size, and structural complexity. Endogenous lipase Lip2p was used as a fusion partner to direct the fused proteins to the extracellular medium. A linker sequence was introduced at the junction of Lip2p and the respective fused protein that contains a hexahistidine tag followed by a TEV protease cleavage site. This allows for a specific and simple purification via IMAC for capturing the secreted proteins from the supernatant followed by a second IMAC for removing all contaminants after proteolytic release of the protein of interest. Up to 174 mg/L fusion protein was obtained using shake flask cultivation. Functionality of each of the purified proteins was confirmed by individual assays. Expression of proteins of interest via Lip2p fusion not only provides a convenient expression and purification scheme but also enables for an online monitoring of accumulation of secreted fusion proteins in the medium by exploiting the intrinsic lipase activity of the fusion.     

Construction of a Chimeric Thermostable Pyrophosphatase To Facilitate Its Purification and Immobilization by Using the Choline-Binding Tag

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.     

Overproduction of single-chain antibodies against human IFN-alpha2B in Escherichia coli and their isolation in biologically active form

The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.     

Engineering proteins for purification.

Over the past decade, a new protein purification technique has emerged as a result of recombinant DNA technology. DNA, encoding additional polypeptide or protein tags, is fused to the gene of interest. Expression of these gene fusions results in protein fusions which may be purified by techniques using the properties of the additional polypeptide tag. This has eliminated the need for extensive screening and optimization procedures previously required for purification.     

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization¹ . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used² . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions³ . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies^1,2.Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding⁴ , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A⁵, a small, bispecific molecule with affinity for both HSA and the novel target was identified⁶ .The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination^1,7.     

Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain-intein tag for purification via a chitin-agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and β-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.     

The refolding, purification, and activity analysis of a rice Bowman-Birk inhibitor expressed in Escherichia coli

A putative rice trypsin/chymotrypsin inhibitor of the Bowman-Birk family, RBBI-8 of about 20 kDa, was expressed in Escherichia coli as a fusion protein bearing an N-terminal (His)6 purification tag. The expressed recombinant protein, rRBBI-8, is insoluble and accumulates as inclusion bodies. The insoluble protein was solubilized in 8 M urea under reducing environment and then refolded into its active conformation under optimized redox conditions. Strategies used to optimize yield and efficiency include selecting the redox system, increasing protein concentration during refolding by adding the denatured protein in a stepwise way, utilizing additives to prevent aggregation, and selecting buffer-exchanging conditions. A Ni-chelate affinity column was then employed to purify the renatured protein. rRBBI-8 shows strong inhibitory activity against trypsin and it can slightly inhibit chymotrypsin. In this study, a refolding and purification system was set up for this cysteine-rich recombinant protein expressed in a prokaryotic system.     

Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus

The spike (S) glycoprotein is one of the major structure proteins of SARS-associated coronavirus (CoV). Fragment 450-650 (S450-650) of the S protein contains receptor-binding domain and neutralizing epitopes. In this study, S450-650 was expressed with a histidine tag in Escherichia coli BL21. Bacterial inclusion bodies containing the recombinant S450-650 were solubilized with 8 M urea and then applied onto a Ni-nitrilotriacetic acid column. On-column refolding and purification was performed. Reduced glutathione and oxidized glutathione were included in the refolding buffer. In the wash and elution buffers, glycerol and glucose were necessary additives to prevent protein aggregation during purification. This refolding and purification procedure allowed production of S450-650 at up to 500 microg/ml in soluble form, which maintained appropriate antigenicity and immunogenicity. It was able to induce strong IgG responses in BALB/c mice. In Western blot assays, the recombinant S450-650 was recognized by monoclonal Ab against the His-tag and also sera from a convalescent SARS patient. S450-650-based ELISA system was able to detect anti-SARS-CoV IgG Abs in patient sera.     

HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification

Over-expression and purification of soluble and functional proteins remain critical challenges for many aspects of biomolecular research. To address this, we have developed a novel protein tag, HaloTag7, engineered to enhance expression and solubility of recombinant proteins and to provide efficient protein purification coupled with tag removal. HaloTag7 was designed to bind rapidly and covalently with a unique synthetic linker to achieve an essentially irreversible attachment. The synthetic linker may be attached to a variety of entities such as fluorescent dyes and solid supports, permitting labeling of fusion proteins in cell lysates for expression screening, and efficient capture of fusion proteins onto a purification resin. The combination of covalent capture with rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional affinity tags and enables efficient capture even at low expression levels. Following immobilization on the resin, the protein of interest is released by cleavage at an optimized TEV protease recognition site, leaving HaloTag7 bound to the resin and pure protein in solution. Evaluation of HaloTag7 for expression of 23 human proteins in Escherichia coli relative to MBP, GST and His₆Tag revealed that 74% of the proteins were produced in soluble form when fused to HaloTag7 compared to 52%, 39% and 22%, respectively, for the other tags. Using a subset of the test panel, more proteins fused to HaloTag7 were successfully purified than with the other tags, and these proteins were of higher yield and purity.     

pH-triggered reversible "stealth" polycationic micelles

Amphiphilic polycations with a "stealth" cationic nature have been designed and synthesized by the PEGylation of polycationic amphiphile via a novel pH responsible benzoic imine linker. The linkage is stable in aqueous solution at physiological pH but cleaves in slight acidic conditions such as the extracellular environment of solid tumor and endosomes. The polymeric micelle formed from the amphiphilic "stealth" polycation contains a pH-switchable cationic surface driven by the reversible detachment/reattachment of the shielding PEG chains due to the cleavage/formation process of the imine linkage. At physiological pH, the micellar surface was shielded by the PEG corona, leading to lower cytotoxicity and less hemolysis, whereas in a mild acidic condition like in endosomes or solid tumors, the deshielding of the PEG chains exposed the positive charge on the micellar surface and retained the membrane disrupting ability. The amphiphilic "stealth" polycation is potentially useful as a drug targeting system toward tumors via endocytosis and trafficked through the endosomal pathway.     

Effects of l-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli

Insulin-like growth factor 1 (IGF1), a therapeutic protein, is highly homologous to proinsulin in 3-dimensional structure. To highly express IGF1 in recombinant Escherichia coli, IGF1 was engineered to be fused with the 6-lysine tag and ubiquitin at its N-terminus (K6Ub-IGF1). Fed-batch fermentation of E. coli TG1/pAPT-K6Ub-IGF1 resulted in 60.8 g/L of dry cell mass, 18% of which was inclusion bodies composed of K6Ub-IGF1. Subsequent refolding processes were conducted using accumulated inclusion bodies. An environment of 50 mM bicine buffer (pH 8.5), 125 mM L-arginine, and 4 °C was chosen to optimize the refolding of K6Ub-IGF1, and 240 mg/L of denatured K6Ub-IGF1 was refolded with a 32% yield. The positive effect of L-arginine on K6Ub-IGF1 refolding might be ascribed to preventing unfolded K6Ub-IGF1 from undergoing self-aggregation and thus increasing its solubility. The simple dilution refolding, followed by cleavage of the fusion protein by site-specific UBP1 and chromatographic purification of IGF1, led production of authentic IGF1 with 97% purity and an 8.5% purification yield, starting from 500 mg of inclusion bodies composed of K6Ub-IGF1, as verified by various analytical tools, such as RP-HPLC, CD spectroscopy, MALDI-TOF mass spectrometry, and Western blotting. Thus, it was confirmed that L-arginine with an aggregation-protecting ability could be applied to the development of refolding processes for other inclusion body-derived proteins.