Fusion tails for the recovery and purification of recombinant proteins.

Several fusion tail systems have been developed to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Tails with a variety of characteristics have been used: (1) entire enzymes with affinity for immobilized substrates or inhibitors; (2) peptide-binding proteins with affinity to immunoglobulin G or albumin; (3) carbohydrate-binding proteins or domains; (4) a biotin-binding domain for in vivo biotination promoting affinity of the fusion protein to avidin or streptavidin; (5) antigenic epitopes with affinity to immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) poly(His) residues for recovery by immobilized metal affinity chromatography; and (8) other poly(amino acid)s, with binding specificities based on properties of the amino acid side chain. Fusion tails are useful at the lab scale and have potential for enhancing recovery using economical recovery methods that are easily scaled up for industrial downstream processing. Fusion tails can be used to promote secretion of target proteins and can also provide useful assay tags based on enzymatic activity or antibody binding. Many fusion tails do not interfere with the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of authentic proteins a site for specific cleavage is often included, allowing removal of the tail after recovery.     

Study of protein splicing and intein-mediated peptide bond cleavage under high-cell-density conditions

Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins.     

The purification of IgY from chicken egg yolk by preparative electrophoresis

Chicken IgY has been purified from egg yolk by preparative electrophoresis on the Gradiflow, a system which has been employed for the purification of a wide range of proteins with high recovery and biological activity. Protein purification on the Gradiflow utilises electrophoresis with selected combinations of porous membranes and buffers. The purification of IgY was achieved by initial PEG lipid precipitation, then a single step Gradiflow run by a strategy based on the relatively high pI range of IgY compared to other egg yolk proteins. The IgY yields obtained from the delipidised supernatant are consistently greater than 80% by immunoassay. The purity of the IgY fraction compared favourably with IgY prepared using three commercial products.     

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.     

Protein purification using a soluble affinity matrix: purification of estrogen receptor with estradiol-polylysine conjugate.

A new strategy for protein purification using a soluble affinity matrix is described. The method was used for purification of estrogen receptor. Cytosols from rat uteri and human fibroid uterine tissue, after fractionation by ammonium sulfate, were treated with estradiol-polylysine conjugate. The highly basic affinity complex was separated from other proteins by DEAE-Sephacel chromatography. After dissociation of the eluted complex with excess estradiol, the receptor was recovered by CM-Sephadex chromatography. A 2000-fold purification of the rat uterine estrogen receptor was obtained with an activity recovery of 35%.     

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.     

COST-EFFECTIVE AND EFFICIENT APPARATUS FOR ELECTROELUTION OF MICRO- AND MACROGRAM QUANTITIES OF PROTEINS FROM POLYACRYLAMIDE GELS

Protein recovery from gel electrophoresis plays a significant role in functional genomics and proteomics. To assist in this, a simple, cost-effective, and efficient apparatus for electroelution of proteins has been designed. The performance of the apparatus was demonstrated using the proteins bovine serum albumin (BSA), phosphorylase, ovalbumin, pepsin, and trypsinogen. In all the cases the yield of elution was found to be consistently greater than 85% and the proteins could be eluted without degradation in less than 15 min. The utility of this method can be extended to protein elution from denatured and native polyacrylamide gels, DNA purification from agarose gels, and oligomeric primers purification from polyacrylamide gels. In addition to this, the method offers an effortless purification and characterization of microbial extracellular proteins. The eluted proteins can be directly used in N-terminal amino acid sequencing, and in amino acid and proteomics analyses.     

Protein recovery from the all-agarose ProSieve gel system.

Electrophoresis in polyacrylamide gels is one of the most powerful tools used for the analysis of proteins. However, this technique is not widely used for protein purification for a variety of reasons such as the following: less than quantitative recoveries; involved, time-consuming methodologies; and impurities in the protein preparations from gel-polymerization by-products that can modify the proteins and interfere with subsequent experiments. As an alternative, we have developed a simple and quantitative recovery procedure for proteins separated by electrophoresis in the all-agarose ProSieve gel system. Using this procedure, greater than 90% of each protein examined was recovered, and these proteins were unaffected by the recovery procedure.     

Cloning,expression and two-step purification of recombinant His-tag enhanced green fluorescent protein over-expressed in Escherichia coli

In this report, we describe a two-step chromatographic procedure for the purification of His-tag EGFP by immobilized metal affinity expanded bed adsorption (IMAEBA) as the capture step and size exclusion chromatography as the polishing step. The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists in purifying recombinant proteins. The procedure described allowed the obtention of 230 mg pure EGFP from 1 l simple batch culture with a recovery of 90%.     

Aqueous two-phase partition of complex protein feedstocks derived from brain tissue homogenates

This study describes the application of aqueous two-phase partition using polyethylene glycol (PEG)-potassium phosphate systems for the direct recovery of proteins, and aggregates thereof, from mammalian brain tissue homogenates. Investigation of established methodologies for the purification of prion proteins (PrP) from bovine brain affected with transmissible spongiform encephalopathy (BSE) has identified an alternative purification regime based on aqueous two-phase partition. This circumvents energy-intensive and rate-limiting unit operations of ultracentrifugation conventionally used for isolation of PrP. Selectivity of various PEG-phosphate systems varied inversely with polymer molecular mass. The maximum protein recovery from bovine brain extracts was obtained with systems containing PEG 300. Manipulation of the aqueous environment, to back-extract protein product from the PEG-rich top phase into the phosphate-rich lower phase, enabled integration of ATPS with conventional hydrophobic interaction chromatography (HIC) which selectively removes obdurate contaminating proteins (i.e. ferritin).     

A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells

The tandem affinity purification (TAP) tag technique has been used with success to identify under nondenaturing conditions protein complexes in yeast. The technique can be used in mammalian cells, but we found that the original technique does not yield enough recovery for the identification of proteins when mammalian cells growing in monolayer have to be used. We present here a modified TAP tag technique that allows sufficient recovery of proteins from mouse fibroblasts growing in monolayer cultures. The recovery allows protein identification by mass spectrometry.     

Efficient recovery of recombinant human erythropoietin from milk of transgenic pigs by two-step pretreatment

The selective removal of impurity proteins and colloidal particles from milk prior to chromatographic purification processes presents a crucial issue in the production of therapeutic proteins from transgenic animals with high recovery yield and purity. We have developed an efficient two-step precipitation method for the recovery of the recombinant human erythropoietin (rhEPO) of interest from transgenic sow milk. Here, rhEPO was partially purified from transgenic sow milk via a two-step precipitation method consisting of ammonium sulfate and divalent metal precipitations, with a yield of approximately 82.1% and a purification fold of 10.4 at a copper concentration of 30 mM. Copper proved to be the strongest flocculating agent among the divalent ions tested for the aggregation of milk proteins under 35%, with ammonium sulfate, zinc, nickel, and calcium demonstrating increasing flocculating capability in the given order. Copper and zinc proved to be appropriate divalent metals for the recovery of rhEPO at high yield and purity, and the optimal concentration ranges of copper and zinc were 20~40 and 40~80 mM, respectively.     

Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.     

Recombinant aprotinin produced in transgenic corn seed: extraction and purification studies

Expression in transgenic plants is potentially one of the most economical systems for large-scale production of valuable peptide and protein products. However, the downstream processing of recombinant proteins produced in plants has not been extensively studied. In this work, we studied the extraction and purification of recombinant aprotinin, a protease inhibitor used as a therapeutic compound, produced in transgenic corn seed. Conditions for extraction from transgenic corn meal that maximize aprotinin concentration and its fraction of the total soluble protein in the extract were found: pH 3.0 and 200 mM NaCl. Aprotinin, together with a native corn trypsin inhibitor (CTI), was captured using a tryspin-agarose column. These two inhibitors were separated using an agarose-IDA-Cu2+ column that proved to efficiently absorb the CTI while the recombinant aprotinin was collected in the flowthrough with purity of at least 79%. The high purity of the recombinant aprotinin was verified by SDS-PAGE and N-terminal sequencing. The overall recombinant aprotinin recovery yield and purification factor were 49% and 280, respectively. Because CTI was also purified, the recovery and purification process studied has the advantage of possible CTI co-production. Finally, the work presented here introduces additional information on the recovery and purification of recombinant proteins produced in plants and corroborates with past research on the potential use of plants as biorreactors.     

Simultaneous refolding/purification of xylanase with a microwave treated smart polymer

Affinity precipitation with a smart polymer, Eudragit S-100 (a methyl methacrylate polymer), was exploited for simultaneous refolding and purification of xylanase. Affinity precipitation consisted of this reversibly soluble-insoluble polymer-binding xylanase selectively. The complex was precipitated by lowering the pH and xylanase was eluted off the polymer using 1 M NaCl. For refolding experiments, the commercial preparation of Aspergillus niger xylanase was denatured with 8 M urea. Addition of microwave irradiated Eudragit S-100 and affinity precipitation led to recovery of 96% enzyme activity by refolding. Simultaneously, the enzyme was purified 45 times. Thermally inactivated preparation, when subjected to similar steps, led to 95% recovery of enzyme activity with 42-fold purification. The strategy has the potential for recovering pure proteins in active forms from overexpressed proteins, which generally form inclusion bodies in E. coli.     

Single step purification of lactoperoxidase from whey involving reverse micelles‐assisted extraction and its comparison with reverse micellar extraction

The extraction of lactoperoxidase (EC 1.11.1.7) from whey was studied using single step reverse micelles‐assisted extraction and compared with reverse micellar extraction. The reverse micelles‐assisted extraction resulted in extraction of contaminating proteins and recovery of lactoperoxidase in the aqueous phase leading to its purification. Reverse micellar extraction at the optimized condition after forward and backward steps resulted in activity recovery of lactoperoxidase and purification factor of the order of 86.60% and 3.25‐fold, respectively. Whereas reverse micelles‐assisted extraction resulted in higher activity recovery of lactoperoxidase (127.35%) and purification factor (3.39‐fold). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) profiles also evidenced that higher purification was obtained in reverse micelles‐assisted extraction as compared of reverse micellar extracted lactoperoxidase. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Recombinant expression and purification of smad proteins

Smads transduce intracellular signals initiated by members of the transforming growth factor beta (TGF beta) family, including activins, TGF betas, and bone morphogenetic proteins. Recently, various models concerning the mechanism of Smad action have been proposed; however, these models are basically qualitative. Quantitative verification of the validity of the models requires significant amounts of purified Smad proteins, but purification of full-length Smad protein has not been straightforward even using recombinant protein expression systems. Here, we report purification of Smad proteins expressed in E. coli as glutathione S-transferase-fused proteins. By glutathione-Sepharose affinity purification, ATP treatment, DEAE-Sepharose and hydroxylapatite columns, expressed Smads were purified to near homogeneity as judged by SDS-PAGE; protein recovery was ca. 1 mg/l culture for Smad2 and 100 microg/l culture for Smad4. The purified Smad proteins had three known in vitro activities: Smad2 phosphorylation by TGF beta receptor complexes immunoprecipitated from COS7 cells, Smad4 binding to Smad-binding DNA element, and Smad2 interaction with calmodulin. The data suggest that purified proteins could be useful for biochemical analyses to evaluate the current models quantitatively.     

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.     

Methods for preparation of low abundance glycoproteins from mammalian cell supernatants

Proteins secreted to mammalian cell supernatants are usually in a low concentration and purity, due to the limitation of the expression systems or the presence of a large amount of contaminant proteins from the cell medium. So, initial protein recovery from cell supernatants requires of a highly specific chromatography step. We compared several purification methods based on affinity chromatography for purification of proteins from cell culture supernatants: metal chelate affinity, strep-tag and immunopurification with a monoclonal antibody. Soluble receptor glycoproteins were engineered with the corresponding peptide tag at their C-terminal end. The proteins were expressed in 293T cells and secreted to the cell supernatant, as monitored by sandwich ELISA. Supernatants were run through the different chromatography columns and several purification-related parameters determined. While all column-retained proteins were easily eluted, the chelating and immunopurification chromatography gave the highest yield and the latest method provided a sample with the highest purity. So, in spite of its cost, immunopurification chromatography gave optimal results for purification of a low abundance protein from a cell supernatant. Finally, we applied a protein expression system together with immunopurification chromatography for preparation of a glycoprotein for crystallization.     

Intracellular production of a soluble and functional monomeric streptavidin in Escherichia coli and its application for affinity purification of biotinylated proteins

Monomeric forms of avidin and streptavidin [(strept)avidin] have many potential applications. However, generation of monomeric (strept)avidin in sufficient quantity is a major limiting factor. We report the successful intracellular production of an improved version of monomeric streptavidin (M4) in a soluble and functional state at a level of approximately 70 mg/L of an Escherichia coli shake flask culture. It could be affinity purified in one step using biotin agarose with 70% recovery. BIAcore biosensor analysis using biotinylated bovine serum albumin confirmed its desirable kinetic properties. Two biotinylated proteins with different degrees of biotinylation (5.5 and 1 biotin per protein) pre-mixed with cellular extracts from Bacillus subtilis were used to examine the use of M4-agarose in affinity purification of protein. Both biotinylated proteins could be purified in high purity with 75-80% recovery. With the mild elution and matrix regeneration conditions, the M4-agarose had been reused four times without any detectable loss of binding capability. The relatively high-level overproduction and easy purification of M4, excellent kinetic properties with biotinylated proteins and mild procedure for protein purification make vital advancements in cost-effective preparation of monomeric streptavidin affinity matrix with desirable properties for purification of biotinylated molecules.