Showing papers on "Affinity chromatography published in 2002"

Elongation factor Tu and E1 β subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae

Abstract: Summary The interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn-binding proteins (FnBPs) were purified to near homogeneity using Fn-coupled Sepharose 4B-affinity column chromatography, and amino acid sequence analysis of the 45 and the 30 kDa proteins identified them as elongation factor Tu (EF-Tu) and pyruvate dehydrogenase E1 β subunit (PDH-B) respectively. The genes for EF-Tu and PDH-B were cloned, and the entire EF-Tu gene and NH2-terminus of PDH-B (NPDH (pyruvate dehydrogenase E1 β subunit from amino acid 1–244)-B) gene were overexpressed in Escherichia coli. The recombinant proteins, rEF-Tu and rNPDH-B, were purified to homogeneity by His-tag affinity column chromatography and used to immunize rabbits. Purified rEF-Tu and rNPDH-B bound to Fn using a ligand immunoblot assay and ELISA. Immunogold electron microscopy with polyclonal antibodies reactive against rEF-Tu (antirEF-Tu) and rNPDH-B (antirNPDH-B) and whole cell radioimmunoprecipitation (WCRIP) revealed the surface location of these proteins. Adherence of viable M. pneumoniae to immobilized Fn was inhibited by antirEF-Tu and antirNPDH-B antisera in a dose-dependent and cumulative manner. These results demonstrate that M. pneumoniae EF-Tu and PDH-B, in addition to their major cytoplasmic biosynthetic and metabolic roles, can be surface translocated, which confers additional important biological functions.

Purification and characterization of a surface protein from Lactobacillus fermentum 104R that binds to porcine small intestinal mucus and gastric mucin.

Abstract: An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.

Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli

Abstract: The adenosine A(2a) receptor belongs to the seven transmembrane helix G-protein-coupled receptor family, is abundant in striatum, vasculature and platelets and is involved in several physiological processes such as blood pressure regulation and protection of cells during anoxia. For structural and biophysical studies we have expressed the human adenosine A(2a) receptor (hA2aR) at high levels inserted into the Escherichia coli inner membrane, and established a purification scheme. Expression was in fusion with the periplasmic maltose-binding protein to levels of 10-20 nmol of receptor per L of culture, as detected with the specific antagonist ligand [(3)H]ZM241385. As the receptor C-terminus was proteolyzed upon solubilization, a protease-resistant but still functional receptor was created by truncation to Ala316. Addition of the sterol, cholesteryl hemisuccinate, allowed a stable preparation of functional hA2aR solubilized in dodecylmaltoside to be obtained, and, increased the stability of the receptor solubilized in other alkylmaltosides. Purification to homogeneity was achieved in three steps, including ligand affinity chromatography based on the antagonist xanthine amine congener. The purified hA2aR fusion protein bound [(3)H]ZM241385 with a K(d) of 0.19 nm and an average B(max) of 13.7 nmol x mg(-1) that suggests 100% functionality. Agonist affinities for the purified solubilized receptor were higher than those for the membrane-bound form. Sufficient pure, functional hA2aR can now be prepared regularly for structural studies.

Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.

Abstract: A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens.

Scalable purification of adeno-associated virus type 2, 4, or 5 using ion-exchange chromatography.

Abstract: The availability of high-titer, high-purity, adeno-associated virus type 2 (AAV2) stocks has dramatically increased our understanding of this virus and its utility as a gene transfer vector. Current methods of purification take advantage of the stable interaction of AAV2 with heparin sulfate. This affinity chromatography, however, is not useful for purifying AAV4 and AAV5, because these serotypes lack heparin-binding activity. We have developed simple ion exchange high-performance liquid chromatography (HPLC) method for purifying different AAV serotypes that does not rely on the affinity of the viruses for heparin. The protocol is fast, efficient, and yields highly infectious material. Analysis of the highly purified virus indicated that more than 90% of the particles contained genomes and were more active than virus purified by cesium chloride (CsCl) gradient purification. This procedure is scalable and can easily be streamlined for large-scale production of recombinant adeno-associated virus (rAAV), regardless of the serotype. Ultimately, the new purification method will further the characterization of rAAV of different serotypes as vectors for gene therapy applications.

Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A.

Abstract: Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. Characterization of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding, with dissociation constants (K(d)) in the range between 0.5 and 3 microm. One variant (Z(IgA1)) showing the strongest binding affinity was further analyzed, and showed that human IgA subclasses (IgA(1) and IgA(2)) as well as secretory IgA were recognized with similar efficiencies. No detectable cross-reactivity towards human IgG, IgM, IgD or IgE was observed. The potential use of the Z(IgA1) affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate, using an affinity column containing a divalent head-to-tail Z(IgA1) affibody dimer construct as a ligand. In addition, efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated.

Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves.

Abstract: Purification of abscisic acid (ABA)-binding proteins is considered to constitute a major step toward isolating ABA receptors. We report here that an ABA-binding protein was for the first time, to our knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional electrophoresis of the purified ABA-binding protein all identified a single protein band with a molecular mass of 42 kD and an isoelectric point 4.86. The Scatchard plot for the purified protein showed a linear function with a maximum binding activity of 0.87 mol mol(-1) protein and an equilibrium dissociation constant of 21 nM, indicating that the purified protein may be a monomeric one, possessing one binding site. The ABA-binding protein was enriched more than 300-fold with a yield of 14%. (-)ABA and trans-ABA were substantially incapable of displacing (3)H-(+/-)ABA bound to the ABA-binding protein, and (+/-)ABA was less effective than (+)ABA in the competition. These findings allow establishment of the stereospecificity of the 42-kD protein and suggest its ABA receptor nature. Pretreatment of the guard cell protoplasts of broad bean leaves with the monoclonal antibody raised against the 42-kD protein significantly decreased the ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.

Purification, characterization, and immunogenicity of the refolded ectodomain of the Plasmodium falciparum apical membrane antigen 1 expressed in Escherichia coli.

Abstract: The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83Gly to 531Glu) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.

Purification and characterization of the recombinant Na+-translocating NADH : quinone oxidoreductase from vibrio cholerae

Abstract: The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), was cloned under the regulation of the PBAD promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, t...

Identification of Escherichia coli outer membrane protein A receptor on human brain microvascular endothelial cells.

Abstract: Neonatal Escherichia coli meningitis continues to be a diagnostic and treatment challenge despite the availability of active antibiotics. Our earlier studies have shown that outer membrane protein A (OmpA) is one of the major factors responsible for Escherichia coli traversal across the blood-brain barrier that constitutes a lining of brain microvascular endothelial cells (BMEC). In this study we showed that OmpA binds to a 95-kDa human BMEC (HBMEC) glycoprotein (Ecgp) for E. coli invasion. Ecgp was partially purified by wheat germ agglutinin and Maackia amurensis lectin (MAL) affinity chromatography. The MAL affinity-purified HBMEC proteins bound to OmpA+ E. coli but not to OmpA− E. coli. In addition, the deglycosylated MAL-bound proteins still interact with OmpA+ E. coli, indicating the role of protein backbone in mediating the OmpA binding to HBMEC. Interestingly, the MAL affinity-bound fraction showed one more protein, a 65-kDa protein that bound to OmpA+ E. coli in addition to Ecgp. Further, the 65-kDa protein was shown to be a cleavage product of Ecgp. Immunocytochemistry of HBMEC infected with OmpA+ E. coli by using anti-Ecgp antibody suggests that Ecgp clusters at the E. coli entry site. Anti-Ecgp antibody also reacted to microvascular endothelium on human brain tissue sections, indicating the biological relevance of Ecgp in E. coli meningitis. Partial N-terminal amino acid sequence of Ecgp suggested that it has 87% sequence homology to gp96, an endoplasmic reticulum-resident molecular chaperone that is often expressed on the cell surface. In contrast, the 65-kDa protein, which could be the internal portion of Ecgp, showed 70% sequence homology to an S-fimbria-binding sialoglycoprotein reported earlier. These results suggest that OmpA interacts with Ecgp via the carbohydrate epitope, as well as with the protein portion for invading HBMEC.

Development of mammalian serum albumin affinity purification media by peptide phage display.

Abstract: Several phage isolates that bind specifically to human serum albumin (HSA) were isolated from disulfide-constrained cyclic peptide phage-display libraries. The majority of corresponding synthetic peptides bind with micromolar affinity to HSA in low salt at pH 6.2, as determined by fluorescence anisotropy. One of the highest affinity peptides, DX-236, also bound well to several mammalian serum albumins (SA). Immobilized DX-236 quantitatively captures HSA from human serum; mild conditions (100 mM Tris, pH 9.1) allow release of HSA. The DX-236 affinity column bound HSA from human serum with a greater specificity than does Cibacron Blue agarose beads. In addition to its likely utility in HSA and other mammalian SA purifications, this peptide media may be useful in the proteomics and medical research markets for selective removal of mammalian albumin from serum prior to mass spectrometric and other analyses.

Biochemical Characterization and Histochemical Localization of Nitric Oxide Synthase in the Nervous System of the Snail, Helix pomatia

Abstract: Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde-sensitive and cytosolic paraformaldehyde-insensitive NADPH-diaphorase. The cytosolic NADPH-diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca2+ and NADPH and was inhibited by NG-nitro-l-arginine (l-NNA). Proteins purified by 2′,5′-ADP affinity chromatography were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60-kDa protein. Characterization of the cDNA of the corresponding 60-kDa NOS-immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca2+- and NADPH-dependent NOS activity, which was partially inhibited by EGTA and l-NNA. Histochemistry showed NADPH-diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH-diaphorase-positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH-diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS-associated/regulating protein in mollusk nervous tissue.

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Abstract: Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.

Anti-idiotypic protein domains selected from protein A-based affibody libraries

Abstract: Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model.

Abstract: Streptococcus parasanguis is a primary colonizer of the tooth surface and plays a pivotal role in the formation of dental plaque. The fimbriae of S. parasanguis are important in mediating adhesion to saliva-coated hydroxylapatite (SHA), an in vitro tooth adhesion model. The Fap1 adhesin has been identified as the major fimbrial subunit, and recent studies suggest that Fap1 is a glycoprotein. Monosaccharide analysis of Fap1 purified from the culture supernatant of S. parasanguis indicated the presence of rhamnose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. A glycopeptide moiety was isolated from a pronase digest of Fap1 and purified by immunoaffinity chromatography. The monosaccharide composition of the purified glycopeptide was similar to that of the intact molecule. The functionality of the glycan moiety was determined using monoclonal antibodies (MAbs) specific for the intact Fap1 glycoprotein. These antibodies were grouped into two categories based on their ability to block adhesion of S. parasanguis to SHA and their corresponding specificity for either protein or glycan epitopes of the Fap1 protein. 'Non-blocking' MAb epitopes were mapped to unique protein sequences in the N-terminus of the Fap1 protein using non-glycosylated recombinant Fap1 proteins (rFap1 and drFap1) expressed in Escherichia coli. In contrast, the 'blocking' antibodies did not bind to the recombinant Fap1 proteins, and were effectively competed by the binding to the purified glycopeptide. These data suggest that the 'blocking' antibodies are specific for the glycan moiety and that the adhesion of S. parasanguis is mediated by sugar residues associated with Fap1.

Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana.

Abstract: The nitrilase AtNIT1 from Arabidopsis thaliana was overexpressed in Escherichia coli with an N-terminal His6 tag and purified by zinc chelate affinity chromatography in a single step almost to homogeneity in a 68% yield with a specific activity of 34.1 U·mg−1. The native enzyme (≈ 450 kDa) consists of 11–13 subunits (38 kDa). The temperature optimum was determined to be 35 °C, and a pH optimum of 9 was found. Thus, recombinant AtNIT1 resembles in its properties the native enzyme and the nitrilase from Brassica napus. The stability of AtNIT1 could be significantly improved by the addition of dithiothreitol and EDTA. The substrate range of AtNIT1 differs considerably from those of bacterial nitrilases. Aliphatic nitriles are the most effective substrates, showing increasing rates of hydrolysis with increasing size of the residues, as demonstrated in the series butyronitrile, octanenitrile, phenylpropionitrile. In comparison with 3-indolylacetonitrile, the rate of hydrolysis of 3-phenylpropionitrile is increased by a factor of 330, and the Km value is reduced by a factor of 23. With the exception of fluoro, substituents in the α position to the nitrile function completely inhibit the hydrolysis.

Matrix metalloproteinase (MMP)-2 and MMP-9 activities in human seminal plasma

Abstract: We report on the existence of two kinds of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, in human seminal plasma. Partial purification of the proteinases was achieved by two steps, consisting of chromatography on a gel-filtration column and then on a gelatin affinity column. Proteinase activities in the chromatography extracts were shown to hydrolyse a fluorescent substrate specific to MMPs (Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2). The proteinases were detected using gelatin-zymography, but were not detected using casein-zymography, and were also inhibited by EDTA, EGTA and o-phenanthroline. Molecular weights of the proteinases were determined by SDS–PAGE, gelatin-zymography and Western blot to be ~92, 84, 72, 67, 52 and 45 kDa. Gelatin-zymography showed three major bands of activity at 72, 67 and 52 kDa and minor bands at 92, 84 and 45 kDa. Apart from the two smallest bands, these proteinases were all recognized by the polyclonal antibodies for MMP-2 or MMP-9. These results indicate that two kinds of pro-form and active-form matrix metalloproteinases, MMP-2 and MMP-9, and their degradation products, are present in human seminal plasma.

Affinity purification of fibrinogen using a ligand from a peptide library.

Abstract: An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.

Isolation and Detection of Human IgA Using a Streptococcal IgA-Binding Peptide

Abstract: Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of approximately 45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA.

Methods for purifying protein

Abstract: A method is provided for separating a protein from one or more other proteins using hydroxyapatite chromatography in which the protein does not bind to hydroxyapatite but the other protein(s) does. In some embodiments, a second protein affixed to a solid support has been used previously to purify the protein by affinity chromatography, and small amounts of the second protein are introduced in the sample during this process. The protein being purified can comprise at least one constant antibody immunoglobulin domain. The second protein can bind to proteins comprising such a domain.