One-step purification of recombinant proteins with the 6xHis tag and Ni-NTA resin (original) (raw)
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Protein Expression and Purification, 2001
Protein affinity tags are widely used for the purifica-We describe the use of the SBP-tag, a new streptavition and detection of recombinant proteins, particularly din-binding peptide, for both the one-step purification from complex mixtures such as lysed cells. However, and the detection of recombinant proteins. The SBPonly a small number of affinity tags are available, and tag sequence is 38 amino acids long and binds to strepthere are significant drawbacks associated with the use tavidin with an equilibrium dissociation constant of of many of them. Commonly used categories of tags, 2.5 nM. We demonstrate that a single-step purification and their limitations, are described below: of SBP-tagged proteins from bacterial extract yields samples that are more pure than those purified using maltose-binding protein or the His-tag. The capacity
Journal of Chromatography A, 1994
The "Strep tag" is a nine amino acid peptide with intrinsic streptavidin-binding activity. If this sequence is genetically fused to the C-terminus of a polypeptide the recombinant protein can be directly purified by affinity chromatography from the host cell extract on immobilized streptavidin. However, variations were observed in the suitability of different commercial streptavidin-agarose preparations for this purpose. Therefore, the influence of the source of streptavidin, the coupling chemistry, and the nature of the affinity chromatography resin was investigated. A procedure was developed for the production of recombinant core streptavidin in Escherichia coli, followed by its efficient refolding and purification with an overall yield of up to 140 mg functional protein per 1 1 bacterial culture. When coupled to activated CH-Sepharose 4B this truncated form of streptavidin showed a performance in the affinity chromatography of Strep tag fusion proteins that was superior to all other combinations tested. In contrast to its conventional preparation from Streptomyces strains the recombinant core streptavidin was produced without a proteolytic processing step. Thus, deleterious effects during the streptavidin affinity purification of proteins due to residual proteolytic activity in the immobilized streptavidin were avoided. The versatility of the optimized purification system was demonstrated with five different Strep tag fusion proteins. * Corresponding author. fore, its removal is not needed for in vitro applications [ 11. Recently, we described the engineering of a C-terminal peptide tag with intrinsic streptavidin-binding activity, which was termed "Strep tag" [2]. This affinity peptide was shown to be suitable for the efficient single-step purification of a bacterially produced, functional antibody F, fragment on immobilized streptavidin using mild competitive elution with biotin or its analogues. Furthermore, the Strep tag was employed for the direct and specific detection of the
Overview of the recombinant proteins purification by affinity tags and tags exploit systems
Journal of Fundamental and Applied Sciences
The advancement in protein expression systems causes yield of each peptide intracellular at least as host. The yield proteins should be purified eventually to provide use possibility or study on them. Therefore rapid and thereby economical purification of an active biological recombinant protein from other cell contents is considered as one of greatest challenges in Biotechnology field. Purification of target protein or enzyme can be facilitated through the integration of genetic sequences with an affinity tag. In this case the tag to the target protein and the protein are expressed as a single unit and the protein can be isolated through one of purification methods by tag from other cellular contents. The affinity tags are commonly used in research laboratories because they can be used to purify wide range of high
Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.
Purification of recombinant proteins by chemical removal of the affinity tag
Applied Biochemistry and Biotechnology, 1998
The efficient removal of a N-or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and a Plasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag in Escherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.
Biotechnology Journal, 2012
Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.
Journal of Molecular Recognition, 2014
In recent years, affinity fusion-tag systems have become a popular technique for the purification of recom- binant proteins from crude extracts. However, several drawbacks including the high expense and low stability of ligands, their leakage during operation, and difficulties in immobilization, make it important to further develop the method. The present work is concerned with the utilization of a ceramic fluorapatite (CFT)-based chromatographic matrix to overcome these drawbacks. A heptapeptide library exhibiting a range of properties have been synthesized and subjected to ceramic fluorapatite (CFT) chromatography to characterize their retention behavior as a function of pH and composition of the binding buffer. The specific binding and elution behavior demonstrates the possible application of CFT-binding peptides as tags for enhancing the selective recovery of proteins by CFT chromatography. To materialize this strategy, a phage-derived CFT-specific sequence KPRSVSG (Tag1) with/without a consecutive hexalysine sequence, KKKKKKKPRSVSG (Tag2), were fused at the C-terminus of an enhanced green fluorescent protein (eGFP). The resulting gene constructs H-eGFP, H-eGFP-Tag1 and H-eGFP-Tag2 were expressed in Escherichia coli strain BL-21, and the clarified cell lysate was applied to the CFT column equilibrated with binding buffer (20–50 mM sodium phosphate, pH 6–8.4). Sodium phosphate (500 mM) or 1 M NaCl in the respective binding buffer was used to elute the fused proteins, and the chromatographic fractions were analyzed by gel electrophoresis. Both the yield and purity were over 90%, demonstrating the potential application of the present strategy.
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Journal of Visualized Experiments, 2012
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 optimization 1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used 2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions 3 . 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 .