Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica (original) (raw)
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Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag
We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model , our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60±0.06 and 1.13±0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports.
Preparative protein purification on underivatized silica
Biotechnology and Bioengineering, 2004
This article describes the use of underivatized silica gel as a preparative stationary phase for process purification of proteins. Although silica has been frequently used as a stationary phase backbone matrix, direct adsorption of proteins on underivatized silica has not been widely exploited for industrial applications. In this study an effort was made to fundamentally understand the interaction mechanisms between a protein and silica surface by using several proteins with a wide range of isoelectric points (pIs) and surface hydrophobicity. Interactions in silica were found to be largely dominated by a combination of ionic and hydrophobic forces. Accordingly, a predictive model was derived for describing linear retention of proteins on silica. Finally, a case study is described investigating the role of silica in an industrial purification process. It was found that the integration of the two modes of interaction confers silica with a unique selectivity that can be very effectively utilized in downstream bioprocessing.
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.
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.
Molecules, 2017
Silica nanoparticles were functionalized with immobilized molecular bait, Cibacron Blue, and a porous polymeric bis-acrylamide shell. These nanoparticles represent a new alternative to capture low molecular weight (LMW) proteins/peptides, that might be potential biomarkers. Functionalized core-shell silica nanoparticles (FCSNP) presented a size distribution of 243.9 ± 11.6 nm and an estimated surface charge of −38.1 ± 0.9 mV. The successful attachment of compounds at every stage of synthesis was evidenced by ATR-FTIR. The capture of model peptides was determined by mass spectrometry, indicating that only the peptide with a long sequence of hydrophobic amino acids (alpha zein 34-mer) interacted with the molecular bait. FCSNP excluded the high molecular weight protein (HMW), BSA, and captured LMW proteins (myoglobin and aprotinin), as evidenced by SDS-PAGE. Functionalization of nanoparticles with Cibacron Blue was crucial to capture these molecules. FCSNP were stable after twelve months of storage and maintained a capacity of 3.1-3.4 µg/mg.
One-step purification of recombinant proteins with the 6xHis tag and Ni-NTA resin
Molecular Biotechnology, 1995
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 440
Journal of Chromatography A, 2003
In this paper, a general procedure is described for the generation of peptide maps of proteins with monolithic silica-based columns. The peptide fragments were obtained by tryptic digestion of various cytochrome c species with purification of the tryptic fragments achieved by reversed-phase high-performance liquid chromatographic methods. Peak assignment of the various peptides was based on evaluation of the biophysical properties of the individual peptides and via mass spectrometric identification. The performance of several different monolithic sorbents prepared as columns of identical cross-sectional dimensions were investigated as part of these peptide mapping studies and the data evaluated by applying solvent strength theory. These studies revealed curvilinear dependencies in the corresponding relative resolution maps. These findings directly impact on the selection of specific sorbent types or column configurations for peptide separations with silica rod monoliths. Moreover, the influence of variations in the amino acid sequence of the cytochrome cs were evaluated with respect to their effect on intrinsic hydrophobicity, the number of experimental observed tryptic cleavage sites, detection limits of the derived fragments in relation to their molecular size, and the chromatographic selectivity and resolution of the various peptides obtained following enzymatic fragmentation of the parent protein. Finally, the scope of these approaches in method development was examined in terms of robustness and efficiency.
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 Chromatography A, 2012
The separation range of superficially porous particles (Fused-core®) has been extended by design of particles with 160 Å pores. These particles show superior kinetics (lower resistance to mass transfer), allowing fast separations of peptides and small proteins (molecular weights of <15,000). The high efficiency and relatively low back pressure of these 2.7 μm fused-core particles has been maintained so that separations can be performed with conventional HPLC instruments. Longer columns can be used for higher resolution of complex mixtures of peptides, such as proteolytic digests. Highly reproducible separations of peptides at elevated temperatures with low pH mobile phases are maintained as a result of a stable bonded stationary phase. The utility of such highly stable materials is exemplified by separations of problematic amyloid peptides at low pH (TFA mobile phase) at an operational temperature of 100 °C. To address the issue of poor peptide peak shape in formic acid-containing mobile phases we show that the addition of 10-20 mM ammonium formate improves peak shape, retention and load tolerance of peptides. Use of the Fused-core particle materials for separations of synthetic peptides and tryptic digests yields peak capacities that are comparable to those obtained using columns packed with sub-2-μm particles, but with less than one-half of the operating back pressure. A peak capacity of 530 was obtained in 150 minutes on coupled columns of HALO Peptide ES-C18 with a combined length of 250 mm.
Analysis of peptides using N -methylpolyvinylpyridium as silica surface modifier for CE-ESI-MS
ELECTROPHORESIS, 2010
In this study, the N-methylpolyvinylpyridinuim polymer has for the first time been used as a silica surface modifier for CE in combination with ESI MS (CE-ESI-MS). The compatibility for ESI-MS was demonstrated by the analysis of peptides and protein digests. The N-methylpolyvinylpyridium surface interacts electrostatically with the ionized silanol groups, giving a cationic surface with a reversed EOF. The surface modifier gave rapid and repeatable separations of peptides, proteins and protein digests at acidic pH for more than 4 h of continuous use. The CE separation yielded peak efficiencies of up to 4.3 Â 10 5 plates/m. The surface coating is highly compatible with ESI and facilitates the separation and analysis of complex peptide mixtures as shown by the analysis of BSA digests.