A novel peptide tag for detection and purification of recombinant expressed proteins

Peptide tags have proven useful for the detection and purification of recombinant proteins. However cross reactions of antibodies raised to the tag are frequently observed due to the presence of host proteins containing all or parts of the tag. In this report we have identified a unique viral peptide sequence, R-tag, that by blast searches is absent from the commonly expression hosts Arabidopsis thaliana, Escherichia coli, Pichia pastoris and mouse myeloma cell NSO. We have prepared monoclonal antibodies to this peptide and confirmed the absence of this peptide sequence from the above genomes by Western blotting. We have also modified protein expression vectors to incorporate this sequence as a fusion tag in expressed proteins and shown its use to successfully purify recombinant proteins by immunoaffinity procedures.     

The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins.

A fusion tag, called FLAG and consisting of eight amino acids (AspTyrLysAspAspAspAspLys) including an enterokinase-cleavage site, was specifically designed for immunoaffinity chromatography. It allows elution under non-denaturing conditions [Bio/Technology, 6 (1988) 1204]. Several antibodies against this peptide have been developed. One antibody, denoted as M1, binds the peptide in the presence of bivalent metal cations, preferably Ca(+). Elution is effected by chelating agents. Another strategy is competitive elution with excess of free FLAG peptide. Antibodies M2 and M5 are applied in this procedure. Examples demonstrating the versatility, practicability and limitations of this technology are given.     

The AviD-tag, a NeutrAvidin/avidin specific peptide affinity tag for the immobilization and purification of recombinant proteins

The widespread success of affinity tags throughout the biological sciences has prompted interest in developing new and convenient labeling strategies. Affinity tags are well-established tools for recombinant protein immobilization and purification. More recently these tags have been utilized for selective biological targeting towards multiplexed protein detection in numerous imaging applications as well as for drug-delivery. Recently, we discovered a phage-display selected cyclic peptide motif that was shown to bind selectively to NeutrAvidin and avidin but not to the structurally similar streptavidin. Here, we have exploited this selectivity to develop an affinity tag based on the evolved DRATPY moiety that is orthogonal to known Strep-tag technologies. As proof of principle, the divalent AviD-tag (Avidin-Di-tag) was expressed as a Green Fluorescent Protein variant conjugate and exhibited superior immobilization and elution characteristics to the first generation Strep-tag and a monovalent DRATPY GFP-fusion protein analogue. Additionally, we demonstrate the potential for a peptide based orthogonal labeling strategy involving our divalent AviD-tag in concert with existing streptavidin-based affinity reagents. We believe the AviD-tag and its unique recognition properties will provide researchers with a useful new affinity reagent and tool for a variety of applications in the biological and chemical sciences.     

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.     

An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins

Determining the composition of messenger ribonucleoprotein (mRNP) particles is essential for a comprehensive understanding of the complex mechanisms underlying mRNA regulation, but is technically challenging. Here we present an RNA-based method to identify RNP components using a modified streptavidin (SA)-binding RNA aptamer termed S1m. By optimizing the RNA aptamer S1 in structure and repeat conformation, we improved its affinity for SA and found a 4-fold repeat of S1m (4×S1m) to be more efficient than the established MS2 and PP7 systems from bacteriophages. We then attached the AU-rich element (ARE) of tumor necrosis factor alpha (TNFα), a well-known RNA motif that induces mRNA degradation, via 4×S1m to a SA matrix, and used the resulting RNA affinity column to purify ARE-binding proteins (BPs) from cellular extracts. By quantitative mass spectrometry using differential dimethyl labeling, we identified the majority of established ARE-BPs and detected several RNA-BPs that had previously not been associated with AREs. For two of these proteins, Rbms1 and Roxan, we confirmed specific binding to the TNFα ARE. The optimized 4×S1m aptamer, therefore, provides a powerful tool for the discovery of mRNP components in a single affinity purification step.     

Separation used for purification of recombinant proteins

The purification of molecules from recombinant cells may be strongly influenced by the molecular biology of gene isolation and expression. At the beginning of the process there may be a demand for information on the minute amounts of proteins and thus for ever increasingly sensitive techniques. Purification of recombinant proteins can differ from conventional purifications in several ways, depending on the solubility of the protein, occurrence in inclusion bodies, creation of fusion proteins with tags that enable simpler purification. Sometimes a (re)naturation step is required to get a bioactive protein. On the other hand, the techniques used in separation are essentially the same as for purification from the natural source and environment.     

Genetic immunization for the recovery and purification of recombinant proteins

A system that uses genetic immunization for recombinant protein recovery and purification is described. The genetic sequence encoding a target protein is subcloned into both a eukaryotic and a prokaryotic vector. With the eukaryotic construct, a rabbit is genetically immunized and specific polyclonal antibodies to the encoded protein raised. The prokaryotic construct is used for bacterial transformation and expression of recombinant protein. Recovery and purification of target recombinant protein are obtained by passing the lysate of expressing bacteria through an immunoaffinity column prepared with the polyclonal antibodies raised in the genetically immunized animal. This method allows purification of recombinant protein without fusion tails and can be applied to purify any protein whose encoding genetic sequence is known.     

A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin AahI.

We created a construct encoding a peptide known to mimic the binding properties of biotin fused to the carboxy-terminus of a scFv fragment that binds a scorpion toxin (AahI). This fusion protein was produced in the periplasm of bacteria and purified to homogeneity by single-step affinity chromatography on streptavidin-agarose with a yield close to 1 mg/l. DNA sequencing, dot blot and mass spectrometric analyses demonstrated the integrity of the soluble immunoconjugate. Fusion to the streptavidin-binding peptide did not affect the ability of the scFv to recognize its antigen with a high affinity (Kd = 2.3 x 10(-10) M). Similarly, the streptavidin-binding property was not impaired in the fusion protein. Thus, the immunoconjugate was bifunctional and had a low molecular mass of 28 kDa. This enabled us to develop rapid and sensitive immunoassays for the specific detection of the toxin AahI accurately to 0.6 ng/ml, opening up new perspectives for the diagnosis of envenomations.     

Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli

Insect cecropins are small basic polypeptides synthesized in fat body and hemocytes in response to bacterial infections or hypodermic injuries. To explore a new approach for high expression of soluble cecropin in Escherichia coli cells, we fused the sequence encoding Musca domestica mature cecropin (named Mdmcec) in-frame to thioredoxin (TRX) gene to construct an expression vector pTRX-6His-Mdmcec. An enterokinase cleavage site was introduced between the 6xHis-tag and Mdmcec to facilitate final release of the recombinant Mdmcec. The fusion protein TRX-6His-Mdmcec was purified successfully by HisTrap HP affinity column and a high yield of 48.0mg purified fusion protein was obtained from 1L culture. Recombinant Mdmcec was readily obtained by enterokinase cleavage of the fusion protein followed by HPLC chromatography, and 11.2mg pure active recombinant Mdmcec was obtained from 1L E. coli culture. The molecular mass of recombinant Mdmcec determined by electrospray ionization-mass spectrometry (ESI-MS) is identical to that of native cecropin. Analysis of recombinant Mdmcec by circular dichroism (CD) indicated that recombinant Mdmcec contained predominantly alpha-helix with some random coil. Antimicrobial activity assays demonstrated that recombinant Mdmcec had a broad spectrum of activity against fungi, Gram-positive and negative bacteria. The procedure described in this study will provide a reliable and simple method for production of different cationic peptides for biological studies.     

Expression,purification and functional characterization of a recombinant scorpion venom peptide BmTXKbeta

BmTXKbeta, a scorpion toxin isolated from the Chinese scorpion Buthus martensii Karsch (BmK), was expressed as a GST fusion protein in BL21 (DE3) strain. The recombinant GST-BmTXKbeta protein was purified by affinity chromatography. When treated with enterokinase, the GST-BmTXKbeta fusion protein released an approximate 6.5kDa protein which was the expected size for correctly processed. About 2mg purified recombinant BmTXKbeta protein (rBmTXKbeta) was produced from 1l bacterial culture, using this expression and purification system. The function of rBmTXKbeta was studied on the rabbit atrial myocyte by whole-cell patch clamp technique. The results showed that rBmTXKbeta inhibited the transient outward current (I(to)) of rabbit atrial myocyte with recovery after washout and the inhibition was concentration-dependent. The rBmTXKbeta prolonged the action potential duration of rabbit atrial myocyte in a concentration-dependent manner, whereas it did not affect the action potential amplitude.     

The development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants

Plants have attracted increasing attention as an expression platform for the production of pharmaceutical proteins due to its unlimited scalability and low cost potential. However, compared to other expression systems, plants accumulate relatively low levels of foreign proteins, thus necessitating the development of efficient systems for purification of foreign proteins from plant tissues. We have developed a novel strategy for purification of recombinant proteins expressed in plants, based on genetic fusion to soybean agglutinin (SBA), a homotetrameric lectin that binds to N-acetyl-D-galactosamine. Previously it was shown that high purity SBA could be recovered from soybean with an efficiency of greater than 90% following one-step purification using N-acetyl-D-galactosamine-agar columns. We constructed an SBA fusion protein containing the reporter green fluorescent protein (GFP) and transiently expressed it in N. benthamiana plants. We achieved over 2.5% of TSP accumulation in leaves of N. benthamiana. Confocal microscopic analysis demonstrated in vivo activity of the fused GFP partner. Importantly, high purity rSBA-GFP was recovered from crude leaf extract with ~90% yield via one-step purification on N-acetyl-D-galactosamine-agar columns, and the purified fusion protein was able to induce the agglutination of rabbit red blood cells. Combined with this, tetrameric assembly of the fusion protein was demonstrated via western blotting. In addition, rSBA-GFP retained its GFP signal on agglutinated red blood cells, demonstrating the feasibility of using rSBA-GFP for discrimination of cells that bear the ligand glycan on their surface. This work validates SBA as an effective affinity tag for simple and rapid purification of genetically fused proteins.     

Apyrase,streptavidin-binding proteins,and antimicrobial activity in Pisum sativum

The 49 kD apyrase (EC 3.6.1.5), streptavidin-binding proteins, and antimicrobial activity in the subcellular fractions from different seed parts of Pisum sativum L. var. Alaska were examined. Except cotyledons, all subcellular fractions contained 49 kD apyrase, and a considerable relationship was found between 49 kD apyrase and NTPase activities that increased with increasing time of germination. The bulk of 49 kD apyrase and NTPase activities was found in the nucleus pellets and cytoskeleton-enriched fraction, indicating their physiological importance. At 72 h of germination, all subcellular fractions of primary stems have a greater amount of 49 kD apyrase and NTPase than primary leaves and much more than primary roots and cotyledonary stalks. All seed parts showed antimicrobial activities, and the bulk of inhibition activities was found in the cytoskeleton-enriched and nucleus pellets, which was greater in the primary stems and leaves than in other parts. Current findings reveal that apyrases have important roles in metabolic activities in all parts of the pea plants except cotyledons. Cotyledons contained much streptavidin-binding proteins, which might have different physiological roles than apyrases.     

The use of recombinant fusion proteases in the affinity purification of recombinant proteins

In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient, and efficient method of affinity purification that can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant "fusion protease" cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step, which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.     

Recovery and purification of plant-made recombinant proteins

Plants are becoming commercially acceptable for recombinant protein production for human therapeutics, vaccine antigens, industrial enzymes, and nutraceuticals. Recently, significant advances in expression, protein glycosylation, and gene-to-product development time have been achieved. Safety and regulatory concerns for open-field production systems have also been addressed by using contained systems to grow transgenic plants. However, using contained systems eliminates several advantages of open-field production, such as inexpensive upstream production and scale-up costs. Upstream technological achievements have not been matched by downstream processing advancements. In the past 10 years, the most research progress was achieved in the areas of extraction and pretreatment. Extraction conditions have been optimized for numerous proteins on a case-by-case basis leading to the development of platform-dependent approaches. Pretreatment advances were made after realizing that plant extracts and homogenates have unique compositions that require distinct conditioning prior to purification. However, scientists have relied on purification methods developed for other protein production hosts with modest investments in developing novel plant purification tools. Recently, non-chromatographic purification methods, such as aqueous two-phase partitioning and membrane filtration, have been evaluated as low-cost purification alternatives to packed-bed adsorption. This paper reviews seed, leafy, and bioreactor-based platforms, highlights strategies for the primary recovery and purification of recombinant proteins, and compares process economics between systems. Lastly, the future direction and research needs for developing economically competitive recombinant proteins with commercial potential are discussed.     

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.     

Enhanced expression,detection and purification of recombinant proteins using RNA stem loop and tandem fusion tags

The creation of a double His-tag fusion that forms a RNA stem loop in the mRNA encoding the N-terminus of the target protein is a novel approach for the enhancement of expression, purification, and detection of a recombinant protein. Compared to a single His-tag fusion, a tandem His-tag fusion RNA stem loop, located downstream of the constitutive groE and Ch promoters, enhanced heterologous gene expression in Brucella, Salmonella, and Escherichia. We demonstrated one-step detection and purification of recombinant green fluorescence protein (GFP) directly from Brucella spp. without using Escherichia coli as an expression host. The amount of purified GFP using the tandem His-tag RNA stem loop increased more than threefold; moreover, the sensitivity of detection increased more than fourfold in comparison to the single His-tag fusion form. This method has the potential to significantly improve heterologous gene expression and high-throughput protein synthesis and purification.     

The purification of recombinant proteins using C-terminal polyarginine fusions

Protein engineering was used to produce urogastrone with a polyarginine fusion. This fusion protein was designed to facilitate purification and illustrates a new approach to protein purification with considerable advantages over conventional techniques.     

Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR

Fibrillar protein aggregates contribute to the pathology of a number of disease states. To facilitate structural studies of these amyloid fibrils by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled peptide are necessary. Bacterial expression of recombinant amyloid proteins and peptides allows uniform isotopic labeling, as well as other patterns of isotope incorporation. However, large-scale production of recombinant amyloidogenic peptides has proven particularly difficult, due to their inherent propensity for aggregation and the associated toxicity of fibrillar material. Yields of recombinant protein are further reduced by the small molecular weights of short amyloidogenic fragments. Here, we report high-yield expression and purification of a peptide comprising residues 11-26 of the Alzheimer's beta-amyloid protein (Abeta(11-26)), with homoserine lactone replacing serine at residue 26. Expression in inclusion bodies as a ketosteroid isomerase fusion protein and subsequent purification under denaturing conditions allows production of milligram quantities of uniformly labeled (13)C- and (15)N-labeled peptide, which forms amyloid fibrils suitable for solid-state NMR spectroscopy. Initial structural data obtained by atomic force microscopy, electron microscopy, and solid-state NMR measurements of Abeta(11-26) fibrils are also presented.     

A calcium-dependent antibody for identification and purification of recombinant proteins

We report a straightforward methodology for purification of recombinant proteins by incorporating a short hydrophilic peptide marker segment at their N-termini. A calcium-dependent antibody that reacts primarily with the first three amino acids of this peptide segment was used to affinity purify the fusion proteins in a single chromatographic step. The marker peptide could subsequently be removed by proteolysis with the enzyme enterokinase.     

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his(6)-tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his(6)-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his(6)-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his(6)-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his(6)-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his(6)-tag.