Immobilization Of Recombinant Proteins Research Articles

Recombinant protein purification and immobilization strategies based on peptides with dual affinity to iron oxide and silica.

Iron oxide and silica-based materials have emerged as attractive protein purification and immobilization matrices. His6 has been reported as an effective affinity tag for both iron oxide and silica. Here, the silica-binding tags CotB1p and Car9 were shown to work as effectively as iron oxide-binding tags. Using EGFP as a model protein, commercially available bare iron oxide (BIONs) or silicon dioxide (BSiNs) nanoparticles as low-cost purification/immobilization matrices, and non-hazardous and mild binding and elution conditions, adsorption and desorption studies were performed with lysates from Escherichia coli-producing cells to compare the performance of these dual-affinity tags. Under the conditions tested, the His6 tag stood out as the best-performing tag, followed by CotB1p. Our findings concluded the promising combination of these tags, BIONs and BSiNs for one-step purification of recombinant proteins, and two-step purification and immobilization of recombinant proteins without intermediate buffer exchange. This proof of concept work set the ground for future evaluation of these purification and immobilization strategies using other proteins with different properties, which will be of interest to expand their utility and applicability. This article is protected by copyright. All rights reserved.

A systematic review about affinity tags for one-step purification and immobilization of recombinant proteins: integrated bioprocesses aiming both economic and environmental sustainability.

The present study reviewed and discussed the promising affinity tags for one-step purification and immobilization of recombinant proteins. The approach used to structure this systematic review was The Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) methodology. The Scopus and Web of Science databases were used to perform the bibliographic survey by which 267 articles were selected. After the inclusion/exclusion criteria and the screening process, from 25 chosen documents, we identified 7 types of tags used in the last 10years, carbohydrate-binding module tag (CBM), polyhistidine (His-tag), elastin-like polypeptides (ELPs), silaffin-3-derived pentalysine cluster (Sil3k tag), N-acetylmuramidase (AcmA tag), modified haloalkane dehalogenase (HaloTag®), and aldehyde from a lipase polypeptide (Aldehyde tag). The most used bacterial host for expressing the targeted protein was Escherichia coli and the most used expression vector was pET-28a. The results demonstrated two main immobilization and purification methods: the use of supports and the use of self-aggregating tags without the need of support, depending on the tag used. Besides, the chosen terminal for cloning the tag proved to be very important once it could alter enzyme activity. In conclusion, the best tag for protein one-step purification and immobilization was CBM tag, due to the eco-friendly supports that can be provided from industry wastes, the fast immobilization with high specificity, and the reduced cost of the process.

Label‐Free Fluorescence Turn‐On Detection of Histidine‐Tagged Proteins Based on Intramolecular Rigidification Induced Emission

AbstractHistidine tag (His‐tag) is a representative epitope tag, which has been widely used in the purification or immobilization of recombinant proteins. In this study, a novel probe CTPE‐IDA‐Ni2+ based on intramolecular rigidification induced emission for fluorescence detecting His‐tagged proteins is successfully developed and experimentally demonstrated. This detecting system is based on TPE derivative incorporated IDA‐Ni2+ moiety for real‐time detection of His‐tagged proteins. In the presence of His‐tagged proteins, the IDA‐Ni2+‐His‐tag complex was formed, which restricted the rotation of two phenyl rings linked with TPE and subsequently resulting in the fluorescence turn on. Results have shown that our probe could directly detect His‐tagged proteins with good selectivity and fast response within 10 min. In addition, we have proved that this system could be used in fluorescent labeling of His‐tagged protein instead of traditional Western blot assay with simple procedure, high quality and low background interference, as well as without aggregation‐caused quenching (ACQ) problems like some organic dyes.

One-step purification and immobilization of recombinant proteins using SpyTag/SpyCatcher chemistry.

Based on the specific and spontaneous formation of isopeptide bonds by SpyCatcher/SpyTag, we have developed a one-step method for purification and immobilization of recombinant proteins. The procedure is to immobilize SpyCatcher on glyoxyl agarose gels, and then the SpyCatcher immobilisate can be used to immobilize the SpyTag-fused protein in the crude extract selectively. A mutant of SpyCatcher (mSC), in which a peptide (LysGlyLysGlyLysGly) was added to the C-terminus of SpyCatcher and three lysine residues around the SpyTag/SpyCatcher binding domain were replaced with arginine, was designed to improve the attachment of SpyCatcher to the support. Compared with wild-type SpyCatcher, mSC can be immobilized on the glyoxyl-agarose support more efficiently, which enables the obtained mSC derivative a high binding capacity of the SpyTag-fused protein. The results showed that the target proteins in the crude enzyme extract were purified and immobilized in one step, and the thermal stability of the immobilized target proteins was also remarkably improved.

Identification of the Domains Involved in Promotion of Silica Formation in Glassin, a Protein Occluded in Hexactinellid Sponge Biosilica, for Development of a Tag for Purification and Immobilization of Recombinant Proteins.

Glassin, a protein occluded in biosilica of the hexactinellid sponge Euplectela, promotes silica formation from silicic acid at room temperature and neutral pH and is characterized by its primary structure which consists of a tandem repeat carrying three distinct domains, histidine and aspartic acid-rich (HD) domain, proline-rich (P) domain, and histidine and threonine-rich (HT) domain. The present study aims to clarify the domain responsible for the promotion of silica formation and to demonstrate usefulness of glassin and its domain as a tag for purification and immobilization of recombinant proteins. When each domain was mixed with silicic acid at neutral pH, silica was formed with HD domain as well as glassin, or a single repeat, but not with P or HT domain. Neither of amino or carboxy-terminal half of HD domain induced silica formation. The amount of silica formed with HD domain was significantly lower than that of glassin or a single repeat. HD domain fused with HT domain raised the amount of silica formed, while a HD domain fused with P domain, a mixture of HD and P domains, or a mixture of HD and HT domains has little effect on the promotion of silica formation. Collectively, a minimum sequence for promotion of silica formation is HD domain, whose activity can be enhanced by HT domain through a topological effect. In addition, practicality of glassin and HD domain was demonstrated by fusion of these sequences to green fluorescent protein which was successfully purified with Ni affinity chromatography and immobilized on silica.

A chemoenzymatic approach enables the site-specific conjugation of recombinant proteins.

Many biotechniques including protein microarray, drug screening, biosensors rely on the immobilization of recombinant proteins on the solid supports. It is well known that random orientation of the immobilized proteins could impair their biologic functions. Thus, it is very important to develop new site-specific immobilization approach. In this study, we presented a chemoenzymatic approach for site-specific conjugation of recombinant proteins onto solid support. In this strategy, the affinity tag on recombinant protein was enzymatically cleaved to expose the N-terminal serine, which was oxidized to carry an aldehyde group and was then covalently coupled to hydrazide resin through hydrazone ligation. As this approach takes advantage of the most frequently used TEV protease, it requires no further sequence design on recombinant protein. This method was validated by site specific coupling of a synthetic peptide and a recombinant protein onto solid supports. It was found that the site specific immobilized SH2 domain is functional and could be used to enrich tyrosine phosphorylated peptides.

Tamavidin, a versatile affinity tag for protein purification and immobilization

Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity and is highly produced in soluble form in Escherichia coli. By contrast, widely used biotin-binding proteins avidin and streptavidin are rarely produced in soluble form in E. coli. In this study, we describe an efficient system for one-step purification and immobilization of recombinant proteins using tamavidin 2 as an affinity tag. A bacterial sialyltransferase and soybean agglutinin were fused to tamavidin 2 and expressed in E. coli and tobacco BY-2 cells, respectively. High-level expressions of the fusion proteins were detected (80 mg l −1 E. coli culture for bacterial sialyltransferase–tamavidin 2 and 2 mg l −1 BY-2 cell culture for soybean agglutinin–tamavidin 2). To immobilize and purify the fusion proteins, biotinylated magnetic microbeads were incubated with the soluble extract from each recombinant host producing the fusion protein and then washed thoroughly. As the result, both fusion proteins were immobilized tightly on the microbeads without substantial loss of activity and simultaneously highly purified (90–95% purity) on the microbeads. Biotin with a longer linker contributed to higher affinity between the fusion protein and biotin. These results suggest that tamavidin fusion technology is a powerful tool for production, purification, and immobilization of recombinant proteins.

Mechanism of interaction of tumor necrosis factor (TNF-a) of macroorganism with Salmonella Enterica cells (Ser. Typhimurium)

The goal of this work was to elucidate the mechanism of direct interaction of bacterial cells with tumor necrosis factor (TNF-alpha; cytokine). It was shown earlier that this interaction facilitated activation of bacterial growth and recultivation of non-cultivated forms in vitro and in vivo. It was shown in experiments with mice deficient in the genes encoding eucaryotic TNF-alpha receptors and infected with salmonella that addition of exogenous TNF-alpha to suspension of infection cells caused a one-day acceleration in the infection start (bacteria planting from spleen) in both knockouted and control mice relative to the same animals infected with the same bacteria without cytokine. Thus, bacteria are able to interact with cytokine even in the absence of eucaryotic receptors. Specificity of the bacterium-cytokine interaction and bacterial protein EF-Tu mediating direct interaction of bacteria with cytokine were identified using the method of immobilization of recombinant protein TNF-alpha-spacer-CSD on cellulose.

Multiple affinity domains for the detection, purification and immobilization of recombinant proteins.

Affinity systems based on specific molecular recognition are valuable tools for detection, purification and immobilization of recombinant proteins. Here, novel multipartite affinity fusion vectors were assembled and investigated to allow flexible binding and elution conditions. The rationale for the assembly of different combinations of affinity domains was to take advantage of the wide variety of molecular interactions of these domains for purification, solubilization, detection and immobilization. In total, seven different affinity tags representing five different types of tag-ligand interactions were studied: (i) monoclonal antibodies-peptides (T7-tag and FLAG peptide); (ii) streptavidin-peptide (Strep-tag); (iii) hexahistidyl-metal ions (His6-tag; (iv) bacterial receptors-serum proteins (staphyloccal protein A-Fc and streptococcal protein G-serum albumin); (v) streptavidin-biotin (in vivo biotinylated peptide). Selected tags were evaluated for the production and purification of Escherichia coli DNA polymerase I (Klenow fragment). On the basis of the results, a vector (pAff2c) was assembled using a novel combination of affinity domains: (i) an in vivo biotinylated peptide; (ii) a His6 sequence, and (iii) a highly soluble serum albumin binding region. Using these three affinities, a wide variety of conditions can be employed for both the binding and the elution steps.

An Escherichia coli plasmid vector system for production of streptavidin fusion proteins: Expression and bioselective adsorption of streptavidin‐β‐galactosidase

Covalently immobilized biotin was used as a biospecific adsorbant to investigate the application of streptavidin as an affinity domain for simultaneous purification and immobilization of recombinant proteins. A streptavidin-beta-galactosidase fusion protein was constructed and tested as a model system. The gene for streptavidin from Streptomyces avidinii was modified by polymerase chain reaction to mutate the stop codon and to facilitate cloning into an Escherichia coli expression vector yielding a versatile plasmid with 37 unique restriction enzyme sites at the 3' end. E. coli beta-galactosidase was cloned in-frame to the streptavidin gene. Analysis of lysates of induced recombinant E. coli cells by SDS-PAGE and Western blots indicated that the 133.6-kDa fusion protein was expressed. Sulfosuccinimidyl-6-(biotinamido) hexanoate was covalently immobilized on 3-aminopropyl-controled-pore glass beads. Exposure of recombinant cell lysates to this support indicated that streptavidin-beta-galactosidase was bioselectively adsorbed. The resulting biocatalyst contained 300 mg protein per gram of beads and exhibited a specific activity of 306 betamol/min per milligram protein with o-nitrophenyl-beta-D-galactopyranoside as substrate corresponding to approximately 50% of that observed for commercially pure E. coli beta-galactosidase.