Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag (original) (raw)
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Engineering in Life Sciences
Silica is widely used for chromatography resins due to its high mechanical strength, column efficiency, easy manufacturing (i.e. controlled size and porosity), and low-cost. Despite these positive attributes to silica, it is currently used as a backbone for chromatographic resins in biotechnological downstream processing. The aim of this study is to show how the octapeptide (RH)4 can be used as peptide tag for high-purity protein purification on bare silica. The tag possesses a high affinity to deprotonated silanol groups because the tag's arginine groups interact with the surface via an ion pairing mechanism. A chromatographic workflow to purify GFP fused with (RH)4 could be implemented. Purities were determined by SDS-PAGE and RP-HPLC. The equilibrium binding capacity of the fusion protein GFP-(RH)4 on silica is 450 mg/g and the dynamic binding capacity around 3 mg/mL. One-step purification from clarified lysate achieved a purity of 93% and a recovery of 94%. Overloading the column enhances the purity to >95%. Static experiments with different buffers showed variability of the method making the system independent from buffer choice. Our designed peptide tag allows bare silica to be utilized in preparative chromatography for downstream bioprocessing; thus, providing a cost saving factor regarding expensive surface functionalization. Underivatized silica in combination with our (RH)4 peptide tag allows the purification of proteins, in all scales, without relying on complex resins.
International Journal of Biological Macromolecules, 2019
Immobilization of protein, compared to the use of free protein, offers improved stability, easy separation and continuous reusability. However, the classic routes for protein immobilization, based on non-specific adsorption, often negatively affect protein functionality. In this study, EctP1 peptide was explored as a novel short fusion tag for non-covalent adsorption on unmodified solid surfaces, silica and titania. A fusion of EctP1 with bovine carbonic anhydrase (BCA) was employed to investigate the optimal binding conditions that could diminish the nonspecific adsorption of Escherichia coli proteins. The stable binding of BCA-EctP1 on titania was observed in the pH range of 2-9, while the stable binding on silica was in the pH range 6-9. Moreover, the immobilized BCA-EctP1 on silica and titania particles showed enhanced thermal and storage stability and retained 95% of its residual activity after 5 uses. We further demonstrated the merits of the noncovalent immobilization of EctP1 fusion proteins to silica and titania in the recovery of the bound proteins. Interestingly, monomeric arginine showed better recovery yield of EctP1 fusion proteins (about 78-84%), compared to the recovery yield by the salts, NaCl and MgCl 2 (about 30-51%). Using BCA and monomeric red fluorescent protein (mRFP) as model proteins, the EctP1 fusion proteins were released in a biologically active form with approximately 80% recovery and 93% purity. Our approach is a simple and reproducible technique for direct immobilization of recombinant proteins from E. coli lysates on solid supports, with the potential high-purity recovery of recombinant proteins.
We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica bio-mineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric L-arginine, at concentrations of 0.3e0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of w70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75e90% recovery with w90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of L-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification.
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.
Highly Specific Dual Enzyme-Mediated Payload Release from Peptide-Coated Silica Particles
Journal of the American Chemical Society, 2010
Stimuli-responsive gate mechanisms offer potential for the controlled passage of payload molecules from a porous carrier vehicle on-demand. We describe a method for the enzyme-mediated release of macromolecular guest molecules from inorganic silica particles coated with a bioactive peptide shell, synthesized precisely by Fmoc chemistry. Specific enzymatic hydrolysis of the peptide shell removes the bulky peptide-terminated Fmoc groups, permitting the selective release of previously entrapped guest molecules.
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.
Coated silica supports for high-performance affinity chromatography of proteins
Journal of Chromatography A, 1989
Polymer-coated silica supports are potentially good stationary phases for high-performance affinity chromatographic separations of proteins. Silica beads have been coated with a polysaccharide (dextran or agarose), substituted by a calculated amount of positively charged diethylaminoethyl functions in order to neutralize the negatively charged silanol groups of silica and to facilitate the formation of a hydrophilic polymeric layer on the inorganic surface. The silica-based supports were prepared in two steps. First, the silica was impregnated with a solution of diethylaminoethylated polymer, and then the coating polymer was crosslinked in order to avoid leakage of the polymeric layer. The supports present minimal non-specific interactions with proteins, as tested by high-performance size-exclusion chromatography. These coated silica supports were coupled with active ligands, such as protein A, concanavalin A and heparin, by conventional coupling methods. The resulting affinity stationary phases were tested by the elution of proteins in order to study their performance in high-performance affinity chromatography.
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.