The derivatization of oxidized polysaccharides for protein immobilization and affinity chromotography

The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5′-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography.     

Large-scale affinity purification — state of the art and future prospects

Enzymes, human plasma proteins and interferon are examples of proteins which are currently being isolated by large scale affinity purification methods. Although affinity chromatography is still the dominant affinity purification technique, others — such as batch affinity adsorption, affinity precipitation and affinity partitioning — offer certain advantages in large-scale work. Immunosorbents based on monoclonal antibodies and affinity adsorbents based on HPLC matrices are expected to be used more frequently on a production scale.     

Aminoalkyl affinity matrices.

Aminoalkyl matrices are used in affinity chromatography of amine oxidases and other proteins with affinity for amino groups. Under appropriate circumstances chromatography on aminoalkyl matrices may yield purification factors around 100 to 1000, and they have been used in affinity purification of many members of the amine oxidase family. Other proteins with affinity for aminoalkyl matrices include thiol ester proteins, lactoferrin, and proteins with lysine-binding kringles (plasminogen, plasminogen activator, apolipoprotein A). The affinity of thiol ester proteins for aminoalkyl matrices is abolished after inactivation of the thiol ester group by reaction with low molecular weight amines including ammonia. Due to this, an ammonium sulphate precipitation step should be included in purification schemes for amine oxidases. The affinity of lactoferrin for aminoalkyl matrices stems from an affinity for the repeating amino groups in glycosaminoglycans, and this explains why lactoferrin requires diamines for efficient elution. The affinity of plasminogen for aminoalkyl groups is exploited in a one-step purification from plasma, and is also utilised in purification schemes for angiostatin, an angiogenesis-inhibiting fragment of plasminogen. Apolipoprotein A is homologous to plasminogen, and also has affinity for aminohexyl columns. The common binding motif for these proteins are lysine-binding kringles. Due to the properties of the amino group itself, aminoalkyl matrices will inevitably also function as anion exchangers, and this must be taken into consideration in the choice of conditions for sample loading, column washing and elution of bound proteins. Depending on the length of the alkyl chain, the matrices also have a potential for hydrophobic interactions. This property has been exploited in the purification of several proteins but must be minimized during affinity chromatography of amine oxidases. In conclusion, aminoalkyl matrices are valuable tools for affinity chromatography of several different proteins, and simple variations of sample pretreatment, sample loading, and column washing and elution conditions allow efficient selective purification of proteins with different affinities for the matrices.     

Cell/adsorbent interactions in expanded bed adsorption of proteins

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidised resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock.     

Histidine ligand affinity chromatography

The sorbents with immobilized histidine as a pseudo affinity ligand with a wide specificity is described. The possibilities and relevant chemistries to use both particulate and flat or hollow fiber membranes as support matrices are discussed. The usefulness of such adsorbents for the purification of a wide variety of proteins, with relevant interaction mechanism are described. Practical protocols of sample quality, capacity and scaled up and scaled down operations are discussed. Possibilities of pyrogen removal from high value blood proteins and their simultaneous recovery in the pure form, using histidine immobilized sorbents are described.     

Expanded bed affinity purification of bacterial α-amylase and cellulase on composite substrate analogue–cellulose matrices

Expanded bed purification of α-amylase and cellulase directly from unclarified fermentation broth was carried out on specially prepared composite affinity matrices. The concept used was incorporation of polymeric substrates/substrate analogue during cross-linking of cellulose to prepare rigid, porous, cross-linked composite affinity matrices for target enzymes. Of the several polymeric substrates/substrate-analogue used, alginic acid (AA) and microcrystalline cellulose (MCC) when used to prepare cross-linked composite matrices with cellulose, resulted in best affinity purification matrices for α-amylase and cellulase, respectively. These matrices were suitable for purification of the enzymes by batch, packed bed as well as expanded bed purification protocols. The optimized expanded bed protocol for α-amylase from Bacillus spp. B3 gave 51-fold purification on AA-CELBEADS with 69% recovery, whereas, cellulase from Bacillus spp. B21 was purified on MCC-CELBEADS to 18-fold purification with 97% recovery. The SDS-PAGE of both purified preparations showed single bands indicating significant purification on composite affinity adsorbents in a single step strategy.     

Novel lectin-like proteins on the surface of human monocytic leukemia cell line THP-1 cells that recognize oxidized cells

Presence of lectin-like receptors on the membranes of human monocytic leukemia cell line THP-1 cells for clustered sialylated poly-N-acetyllactosaminyl sugar chains on the membranes of oxidized erythrocytes and T-lympoid cells was investigated. Membranes of THP-1 cells differentiated into macrophages were solubilized, and the membrane proteins obtained by affinity chromatographies using lactoferrin-Sepharose and band 3-Sepharose were purified by successive DE column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins of 50, 60, and 80 kDa with specificity to bind to sialylated poly-N-acetyllactosaminyl sugar chains were detected in the chromatographic fractions. A 50-kDa protein was isolated in a pure form. N-Terminal amino acid sequence of the protein was Lys-Gln-Lys-Val-Ala-Gly-Lys-Gln-Pro-Val-, which has not been found in the N-terminal regions of the hitherto known proteins. The antibody, raised against the chemially synthesized peptide composed of the N-terminal amino acid sequence, bound to 50-, 60-, and 80-kDa proteins as analyzed by immunoblotting and immunoprecipitation, indicating that these proteins had the same N-terminal amino acid sequence. The results demonstrate that THP-1 cells have novel 50-, 60-, and 80-kDa lectin-like proteins with the same N-terminal amino acid sequence on the cell surface which would bind to clustered sialylated poly-N-acetyllactosaminyl sugar chains generated on oxidized erythrocytes and T-lymphoid cells.     

Studies on endotoxin removal mechanism of adsorbents with amino acid ligands

In this paper, a series of adsorbents with different amino acid ligands for endotoxin removal were prepared and endotoxin adsorption capacities (EAC) in aqueous solution were studied using an affinity column. The results showed that the property and structure of amino acid ligands have great influence on EAC. As the increasing of isoelectric point and polarity of amino acids ligands, EACs of the adsorbents increased. In addition, computer simulation method was employed to a further investigation on the interaction between endotoxins and ligands. Based on the results, some adsorbents were applied to remove endotoxin from endotoxemia rabbit's serum. Similar adsorption results were observed and the removal efficiency of adsorbents with Arg, Ser ligands is up to 78%.     

Pseudoproteoglycan (pseudoPG) probes that simulate PG macromolecular structure for screening and isolation of PG-binding proteins

A proteoglycan (PG) monomer is a macromolecule consisting of one or more glycosaminoglycan (GAG) chains attached to a core protein. PGs have signaling roles and modulatory functions in the extracellular matrix and at the cell surface. To elucidate the functions of higher-order PG structures, pseudoPGs that imitate the PG structure were prepared to develop probes and affinity adsorbents. Poly-l-lysine (PLL) or polyacrylamide (PAA) was coupled with various GAGs, then biotinylated, and the remaining amino groups were blocked to obtain the pseudoPG probes, biotinyl PLL (BPL)- or PAA (BPA)-GAGs. Lactoferrin exhibited 30-times higher affinity toward BPL-heparin than the conventional single-strand probe, biotin-hydrazide-heparin. Heparin-PLL was immobilized on a formyl-Sepharose and compared with the Hep-Sepharose in which heparin was directly immobilized to amino-Sepharose. Screening for ligands in normal rat brain revealed several proteins that specifically bound to either of the two adsorbents, indicating that the heparin-binding proteins exhibit specific recognition depending on the higher-order structure of the PG.     

Challenges and opportunities in the purification of recombinant tagged proteins

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.     

A small bispecific protein selected for orthogonal affinity purification

A novel protein domain with dual affinity has been created by randomization and selection. The small alkali-stabilized albumin-binding domain (ABD*), used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost a quarter of its amino acids, a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z2 domain/protein A was successfully selected using phage display. Moreover, the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for affinity purification on two different matrices has been investigated using the two ligands, the HSA matrix and the protein A-based, MabSelect SuRe matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification using two different matrices was created. Successful purification of all three versions was efficiently carried out using this strategy.     

Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells

A new matrix for affinity chromatography using pteroylglutamic acid coupled to an epoxy-activated matrix via hexanediamine resulted in negligible ligand leakage and permitted the purification of soluble and membrane-associated folate-binding proteins from human leukemia cells contained in a human spleen. Two species of membrane-associated folate-binding proteins were purified from the solubilized membrane fraction of the tissue using 2 M guanidine-HCl to elute the proteins from the affinity matrix. The higher molecular weight binding protein had an Mr of approximately 310,000 and the smaller species had an Mr of approximately 28,000 by gel filtration. By SDS-polyacrylamide gel electrophoresis the smaller species of membrane-associated protein had a molecular weight of 35,500, but the molecular weight of the larger membrane-associated species could not be determined by this method because of the high concentration of residual Triton X-100 in the sample which interfered with the silver staining of the gel. Two folate-binding proteins, which by SDS-polyacrylamide gel electrophoresis had molecular weights of 34,500 and 32,000, were purified from the 44,000 X g supernatant fraction of the tissue homogenate by acid elution from the affinity matrix. Despite the different cell components from which the soluble and membrane-associated folate-binding proteins were purified, the amino acid compositions were similar, especially with respect to the apolar amino acids. All these forms of folate-binding proteins had higher affinity for oxidized than for reduced folates, and very low affinity for 5-formyltetrahydrofolate and methotrexate. Although these proteins cross-react with one antiserum raised previously to a folate-binding protein from other human leukemia cells, they do not cross-react with the folate-binding proteins purified from two other sources of human leukemia cells, from human placenta, or from the human KB cell line.     

Selective oxidation of methionine residues in proteins.

Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).     

Use of affinity matrices in determining steric requirements for substrate binding: binding of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase from Salmonella typhimurium to Sepharose-Anthranilate derivatives

The binding of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT) in crude extracts of Salmonella typhimurium to Sepharose-anthranilic acid derivatives has been studied. When anthranilic acid was covalently bound to hydrocarbon “arms” by its free amino group or phenyl moiety, the derivatives thus prepared bound PRT preferentially. PRT activity could be eluted from these adsorbents using buffers of increasing pH. The specific activity of PRT eluted from these Sepharose-anthranilate adsorbents was 10- to 12-fold that of the original extract. Anthranilic acid bound to aminohexamethylimino-Sepharose by its free carboxyl group did not bind PRT effectively. Results indicated that the carboxyl group of anthranilic acid must be free in order for PRT to bind the Sepharose-anthranilate derivatives. Throughout this study, control columns consisting of Sepharose derivatives containing the appropriate hydrocarbon “arms” alone were used to investigate the possibilities of nonspecific enzyme binding. The findings obtained stress the necessity of using such controls in any affinity studies of this type. The prospect of utilizing the techniques of affinity chromatography to examine the requirements for the binding of proteins to specific ligands is discussed.     

Interaction of oxidized glutathione with allyl isothiocyanate

Isothiocyanates formed from glucosinolates in Brassica species have a strong affinity for amino acids and proteins, especially for their thiol, sulphide and terminal amino groups. To investigate the action of isothiocyanate on cystine residues in proteins and peptides, the present study on the interaction between allyl isothiocyanate and oxidized glutathione under physiological conditions was undertaken. Oxidized glutathione was oxidatively cleaved to some modified glutathiones by the attack of allyl isothiocyanate on its disulphide bond. Two new modified products were isolated from the reaction mixture by gel chromatography and HPLC, and their structures were determined by NMR and mass spectral analyses as glutathionyl N-allyldithiocarbaramate and its allyl thiohydantoin derivative. The formation of these products indicated oxidative cleavage of the disulphide bond in the cystine residue; the electrophilic attack of the isothiocyanate on the sulphur atom must cleave the disulphide bond oxidatively to dithiocarbamate and sulphenate, as in the case of cystine.     

Biosynthetic incorporation of oxidized amino acids into proteins and their cellular proteolysis

We demonstrate that oxidized amino acids can be incorporated into proteins by protein synthesis. The level of incorporation into protein was dependent on the concentration of oxidized amino acid supplied to the cells. At low levels of incorporation, the oxidized amino acids examined increased the degradation rate of the cell proteins. Degradation of certain proteins containing high levels of DOPA (but not ortho or meta tyrosine) was decreased to below the basal degradation rates suggesting that DOPA may contribute to proteins becoming resistant to proteolysis. Changes in the degradation rates of the oxidized amino acid-containing proteins was shown to have no impact on the degradation rates of native proteins, indicating that the activity of the degradative machinery was not affected. We demonstrate that oxidized proteins are selectively degraded by the proteasomes and provide evidence to suggest that the proteasomes and the endosomal-lysosomal systems may act in sequence as well as in parallel. The incorporation approach, unlike cell studies in which an exogenous oxidant is used, allows the degradation rates of the oxidatively modified proteins to be selectively measured, offering a greater sensitivity as well as greatly reducing toxicity to the cell and avoiding oxidative modification of other cell components.     

Development of novel yeast cell surface display system for homo-oligomeric protein by coexpression of native and anchored subunits

Streptavidin derived from Streptomyces avidinii was displayed on the cell surface of the yeast Saccharomyces cerevisiae by cell-surface engineering using two types of plasmid for the expression of a native subunit and an anchored subunit fused with the C-terminus of 318 amino acids of Flo1p containing a glycosylphosphatidylinositol anchor attachment signal. The displayed streptavidin had the binding ability for biotinylated compounds. This was confirmed by fluorescence microscopy after the adsorption of yeast cells displaying streptavidin and biotinylated fluorescein isothiocyanate. On the other hand, streptavidin produced by cells harboring only the plasmid for the expression of the anchored subunit showed a very low binding activity for biotinylated compounds. Cells displaying streptavidin may constitute novel whole-cell affinity adsorbents widely used for immunoassay and biosensing. This coexpression method will ensure that proteins, such as homo- and hetero-oligomeric proteins, are displayed on the cell surface in an active form.     

Method to site-specifically identify and quantitate carbonyl end products of protein oxidation using oxidation-dependent element coded affinity tags (O-ECAT) and nanoliquid chromatography Fourier transform mass spectrometry

Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. This presents a significant advancement over current methods, combining strengths of current methods and adding the abilities to multiplex, quantitate, and probe more modified amino acids. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1,4,7,10-tetraazacyclododecane-N,N',N' ',N'-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. O-ECAT can be loaded with a variety of metals, which yields the ability to generate mass pairs and multiplex multiple samples. The O-ECAT moiety also serves as a handle for identification, quantitation, and affinity purification. After proteolysis, the AOD-tagged peptides are affinity purified and analyzed by nanoLC-FTICR-MS (nanoliquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry), which provides high specificity in extracting coeluting AOD mass pairs with a unique mass difference and allows relative quantitation based on isotopic ratios. Using this methodology, we have quantified and mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation both at the protein and amino acid levels. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic, and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure, validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities toward oxidation, independent of amino acid residue. This novel methodology not only has the ability to identify and quantitate oxidized proteins but also yields site-specific quantitation on a variety of individual amino acids. We expect to extend this methodology to study disease-related oxidation.     

The construction of amino acid substitution matrices for the comparison of proteins with non-standard compositions

MOTIVATION: Amino acid substitution matrices play a central role in protein alignment methods. Standard log-odds matrices, such as those of the PAM and BLOSUM series, are constructed from large sets of protein alignments having implicit background amino acid frequencies. However, these matrices frequently are used to compare proteins with markedly different amino acid compositions, such as transmembrane proteins or proteins from organisms with strongly biased nucleotide compositions. It has been argued elsewhere that standard matrices are not ideal for such comparisons and, furthermore, a rationale has been presented for transforming a standard matrix for use in a non-standard compositional context. RESULTS: This paper presents the mathematical details underlying the compositional adjustment of amino acid or DNA substitution matrices.