Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

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 optimization¹ . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used² . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions³ . 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.Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding⁴ , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A⁵, a small, bispecific molecule with affinity for both HSA and the novel target was identified⁶ .The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination^1,7.     

Improving the tolerance of a protein a analogue to repeated alkaline exposures using a bypass mutagenesis approach

Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

Analysis and use of the serum albumin binding domains of streptococcal protein G

Streptococcal protein G is an IgG-binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG-binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG- and albumin-binding, different parts of the receptor molecule were produced in E. coli using a coupled expression/secretion system. Affinity chromatography, using IgG or HSA immobilized on Sepharose, showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin-binding. The structure of this part of the protein suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one-step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification is discussed.     

Essential role of the concentration of immobilized ligands in affinity chromatography:: Purification of guanidinobenzoatase on an ionized ligand

Guanidinobenzoatase, a plasma protein with possible application as a ‘tumor marker’, has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by ‘controlled’ immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 μmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 μmol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, ×220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.     

Membrane-spanning domain of bovine foamy virus transmembrane protein having cytotoxicity

Foamy viruses (FVs) have broad cellular tropism infecting vertebrates from fish to human being,which indicates that Env protein has a high capability for membrane fusion.Conservative features in all FV transmembrane (TM) proteins include a region of hydrophobic domain called membrane-spanning domain (MSD),which contains several stretches of hydrophobic amino acids.To investigate whether these features were associated with the cytotoxicity effect of TM on Escherichia coli,a series of mutants were constructed and expressed in the E.coli BL21 (DE3) using pET-32a (+) as expressing vector.The results showed that only TM3 without MSD was expressed in E.coli,whereas the other two containing full or part of the MSD (TM1 and TM2) could not be expressed.Furthermore,the bacterial amount and living bacteria analysis revealed that the cytotoxicity of TM was dependent on its MSD,especially on the stretches of hydrophobic amino acids.Western blotting analysis showed that TM3 protein was purified with affinity purification.     

Protein A chromatography for antibody purification

Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its affinity to immunoglobulins, SPA has found widespread use as a tool in the detection and purification of antibodies and the molecule has been further developed to one of the most employed affinity purification systems. Interestingly, a minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications. This review will focus on the development of different affinity molecules and matrices for usage in antibody purification.     

Affinity chromatography of plasma proteins (guanidinobenzoatase): use of mimetic matrices and mimetic soluble ligands to prevent the binding of albumin on target affinity matrices

Serum albumin is the most abundant protein in plasma and it has a high capacity to bind many small compounds and macromolecules. In this way, albumin may promote important interferences during affinity chromatography of plasma proteins. Guanidinobenzoatase (GB) is a very relevant plasma protease that seems to be related to tumoral processes. This enzyme may be adsorbed on tailor-made agmatine-amide-agarose (CH-A) supports (e.g., the ones having 2 μmol of guanidino groups per ml of agarose attached to the support, through a 6 C aliphatic chain). Such tailor-made supports containing a very low concentration of ionized groups are hardly able to adsorb any protein by anion-exchange. However, they are able to strongly adsorb albumin. In order to solve this problem new mimetic affinity matrices have been designed: (i) by using the same ligand immobilized through a different chemical linkage [guanidino groups attached via secondary amino bonds, (AEA)] or (ii) by using slightly different ligands (e.g., 1,8-octanediamine containing a primary amino group instead of a guanidino one) also attached to the support via amido bonds (CH-DAO). Albumin adsorbs on the target and on the two mimetic matrices while GB is mainly adsorbed on the target one. Moreover, the adsorption of albumin on the affinity matrix (CH-A) is very strongly inhibited by the presence of low concentrations of soluble ligands (e.g., 1,8-octanediamine containing two ionized primary amino groups). On the contrary, the adsorption of GB on CH-A is hardly inhibited by the presence of such mimetic soluble ligand. In this way, the former offering of crude GB samples to AEA plus the use of mimetic inhibitors during adsorption of the extract on CH-A completely prevent the undesirable adsorption of albumin. In a such way, an extremely selective adsorption of GB can be performed. Such an improved chromatography procedure allows a very easy affinity purification and detection of GB.     

Investigation of Human Albumin-Induced Circular Dichroism in Dansylglycine

Induced circular dichroism (ICD), or induced chirality, is a phenomenon caused by the fixation of an achiral substance inside a chiral microenvironment, such as the hydrophobic cavities in proteins. Dansylglycine belongs to a class of dansylated amino acids, which are largely used as fluorescent probes for the characterization of the binding sites in albumin. Here, we investigated the ICD in dansylglycine provoked by its binding to human serum albumin (HSA). We found that the complexation of HSA with dansylglycine resulted in the appearance of an ICD band centred at 346 nm. Using this ICD signal and site-specific ligands of HSA, we confirmed that dansylglycine is a site II ligand. The intensity of the ICD signal was dependent on the temperature and revealed that the complexation between the protein and the ligand was reversible. The induced chirality of dansylglycine was susceptive to the alteration caused by the oxidation of the protein. A comparison was made between hypochlorous acid (HOCl) and hypobromous acid (HOBr), and revealed that site II in the protein is more susceptible to alteration provoked by the latter oxidant. These findings suggest the relevance of the aromatic amino acids in the site II, since HOBr is a more efficient oxidant of these residues in proteins than HOCl. The three-dimensional structure of HSA is pH-dependent, and different conformations have been characterised. We found that HSA in its basic form at pH 9.0, which causes the protein to be less rigid, lost the capacity to bind dansylglycine. At pH 3.5, HSA retained almost all of its capacity for binding to dansylglycine. Since the structure of HSA at pH 3.5 is expanded, separating the domain IIIA from the rest of the molecule, we concluded that this separation did not alter its binding capacity to dansylglycine.     

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.     

Biomimetic design of affinity peptide ligands for human IgG based on protein A-IgG complex

Staphylococcal protein A (SpA) has been widely used as an affinity ligand for the purification of immunoglobulin G (IgG). Based on the affinity motif of SpA, we have herein developed a biomimetic design strategy for affinity peptide ligands of IgG. First, according to the distribution of the six hot spots of the SpA affinity motif determined previously, the number of residues that should be inserted into between the hot spots was determined. Cysteine was introduced as one of the middle inserted residues of the peptide for later immobilization. Then, amino acid location was performed to identify other amino acid residues for insertion, leading to the construction of a peptide library. The library was screened by using different molecular simulation protocols, resulting in the selection of 15 peptide candidates. Thereafter, molecular dynamics simulations were performed to validate the dynamics of the affinity interactions between the candidates and IgG, and 14 of them were found to keep high affinities. Finally, the affinity and specificity of the top one ligand FYWHCLDE were exemplified by protein chromatography and IgG purification. The results indicate that the design strategy was successful and the affinity peptide ligand for IgG is promising for application in antibody purifications.     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine

Fusion of peptide‐based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram‐range amounts of proteins. IMAC‐Ni(II) columns have become the natural partners of 6xHis‐tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His‐tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur‐containing molecules. In this work, we evaluated two different cysteine‐ and histidine‐containing six amino acid tags linked to the N‐terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine‐containing tagged GFPs were able to bind to IMAC‐Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC‐Ni(II) system reaches less than 20% recovery of the cysteine‐containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC‐Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.     

Purification of human IgG by negative chromatography on ω-aminohexyl-agarose

The ω-aminohexyl diamine immobilized as ligand on CNBr- and bisoxirane-activated agarose gel was evaluated for the purification of human immunoglobulin G (IgG) from serum and plasma by negative affinity chromatography. The effects of matrix activation, buffer system, and feedstream on recovery and purity of IgG were studied. A one-step purification process using Hepes buffer at pH 6.8 allowed a similar recovery (69–76%) of the loaded IgG in the nonretained fractions for both matrices, but the purity was higher for epoxy-activated gel (electrophoretically homogeneous protein with a 6.5-fold purification). The IgG and human serum albumin (HSA) adsorption equilibrium studies showed that the adsorption isotherms of IgG and HSA obeyed the Langmuir–Freundlich and Langmuir models, respectively. The binding capacity of HSA was high (210.4 mg mL^?1 of gel) and a positive cooperativity was observed for IgG binding. These results indicate that immobilizing ω-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma.     

Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.     

Chemical synthesis and evaluation of a backbone‐cyclized minimized 2‐helix Z‐domain

The Z‐molecule is a small, engineered IgG‐binding affinity protein derived from the immunoglobulin‐binding domain B of Staphylococcus aureus protein A. The Z‐domain consists of 58 amino acids forming a well‐defined antiparallel three‐helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three‐helix bundle. The small size and the stable three‐helix structure are two attractive properties comprised in the Z‐domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein‐based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z‐domain and joining the N‐ and C‐termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two‐helix Z‐domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N‐ and C‐termini. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of stearic acids on resveratrol-HSA interaction

The interaction between the natural polyphenol resveratrol and human serum albumin (HSA), the most abundant transport protein in plasma, has been studied in the absence and in the presence of up to six molecules of stearic acids (SA) pre-complexed with the protein. The study has been carried out by using the intrinsic fluorescence of both HSA and resveratrol. Protein and polyphenol fluorescence data indicate that resveratrol binds to HSA with an association constant k _a ?=?(1.10?±?0.14)?×?10⁵?M^?1 and (1.09?±?0.02)?×?10⁵?M^?1, respectively, whereas Job plot evidences the formation of an equimolar protein/drug complex. Low SA content associated with HSA does not affect significantly the structural conformation of the protein and its interaction with resveratrol, whereas high SA content induces conformational changes in the protein, and reduces resveratrol binding affinity. The photostability of resveratrol in the different samples changes in the order: buffer <?(high [SA]/HSA)?<?HSA?<?(low [SA]/HSA). The results on (SA/HSA)-resveratrol samples highlight the ability of the protein to bind hydrophobic and amphiphilic ligands and to protect from degradation an important antioxidant molecule under biologically relevant conditions.