Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications (original) (raw)
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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
Protein Expression and Purification, 2009
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 equilibriumbased 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 His 6 Tag 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.
Journal of Molecular Recognition, 2001
In this article, recent progress related to the use of different types of polypeptide fusion handles or 'tags' for the purification of recombinant proteins are critically discussed. In addition, novel aspects of the molecular cassette concept are elaborated, together with areas of potential application of these fundamental principles in molecular recognition. As evident from this review, the use of these concepts provides a powerful strategy for the high throughput isolation and purification of recombinant proteins and their derived domains, generated from functional genomic or zeomic studies, as part of the bioprocess technology leading to their commercial development, and in the study of molecular recognition phenomena per se. In addition, similar concepts can be exploited for high sensitivity analysis and detection, for the characterisation of protein bait/ prey interactions at the molecular level, and for the immobilisation and directed orientation of proteins for use as biocatalysts/biosensors.
Journal of Chromatography A, 2016
A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11 mg ml −1 and 0.48 mg ml −1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M −1 affinity constants and Qmax values of 19.11 ± 2.60 ug g −1 and 79.39 ug g −1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents.
Fusion Tag Design Influences Soluble Recombinant Protein Production in Escherichia coli
International Journal of Molecular Sciences
Fusion protein technologies to facilitate soluble expression, detection, or subsequent affinity purification in Escherichia coli are widely used but may also be associated with negative consequences. Although commonly employed solubility tags have a positive influence on titers, their large molecular mass inherently results in stochiometric losses of product yield. Furthermore, the introduction of affinity tags, especially the polyhistidine tag, has been associated with undesirable changes in expression levels. Fusion tags are also known to influence the functionality of the protein of interest due to conformational changes. Therefore, particularly for biopharmaceutical applications, the removal of the fusion tag is a requirement to ensure the safety and efficacy of the therapeutic protein. The design of suitable fusion tags enabling the efficient manufacturing of the recombinant protein remains a challenge. Here, we evaluated several N-terminal fusion tag combinations and their inf...
Biotechnology Progress, 2004
The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein. Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellullose resin Avicel PH101 and retains the biological activity of the fusion partner. Affinity purification of protein A-CBM2a from the supernatant of a recombinant E. coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 (3. Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions. Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein.
An orthogonal fusion tag for efficient protein purification
Methods in molecular biology (Clifton, N.J.), 2014
Protein fusion tags are important tools in research when robust methods for protein purification and detection are required. In this chapter we present an efficient method for stringent protein purification. A small domain, denoted ABDz1, with affinity for both human serum albumin and Protein A has been developed. The purification tag is based on an albumin-binding domain from Streptococcal Protein G that was engineered to bind Protein A. The ABDz1-tag can be fused to any protein of choice and the purification can be performed using standard laboratory equipment. In this chapter a method for purification of ABDz1-tagged proteins using two successive affinity purification steps is described.
A novel peptide tag for detection and purification of recombinant expressed proteins
Protein Expression and Purification, 2007
Peptide tags have proven useful for the detection and puriWcation 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 iden-tiWed 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 conWrmed the absence of this peptide sequence from the above genomes by Western blotting. We have also modiWed protein expression vectors to incorporate this sequence as a fusion tag in expressed proteins and shown its use to successfully purify recombinant proteins by immuno-aYnity procedures.