Albumin affinity tags increase peptide half-life in vivo

Small organic molecules that bind tightly to serum albumin were applied to the amino terminus of an anticoagulant peptide in an effort to increase its protein binding in vivo. The tagged peptides were evaluated for their ability to be retained on liquid chromatographic columns with serum albumins incorporated into the stationary phase. Those which demonstrated significant affinity were administered intravenously to rabbits and found to have significantly increased plasma half-lives. Novel affinity tags were identified by appending a focused library of compounds to a model tetrapeptide and evaluating the resulting compounds' ability to bind to the serum albumin columns. The most promising were synthesized as the full length peptides and again evaluated in vivo. They were found to have still longer half-lives than the first generation compounds.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.     

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.     

A high-affinity zinc-binding plasma protein in channel catfish (Ictalurus punctatus).

1. Channel catfish (Ictalurus punctatus) have a remarkably high concentration of zinc (Zn) in their blood serum, about 20 micrograms/ml. However, compared to mammals, the concentrations of Zn in their tissues are not remarkable. The serum Zn is dialyzable against a solution containing 1 mM EDTA. 2. Following separation of serum proteins by gel-filtration most of the Zn was recovered in a fraction with the same peak volume of elution for the Zn and protein concentrations and having a molecular weight similar to bovine serum albumin. 3. Binding of Zn to such sites was not changed by Cu2+, Cd2+, Ca2+, or La3+. N-ethylemaleimide (NEM) appeared to decrease binding slightly. 4. Equilibrium dialysis with a Scatchard plot analysis of these data suggested that a single set of binding sites was present on the protein(s) with KD of 2 x 10(-5) M. There were binding sites for 35 x 10(-8) M Zn/mg protein. 5. Parallel equilibrium dialysis measurements using human, rabbit and chicken albumins indicated that the catfish serum protein(s) had a much higher affinity and binding capacity for Zn than the albumins in these species. 6. The catfish Zn serum-binding protein may be an albumin. The possible physiological significance of such a serum protein in freshwater fish is discussed.     

The antibody–antigen interaction at an aqueous–solid interface: A study by means of polarized infrared ATR spectroscopy

The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit > bovine ? ovalbumin; with anti-ovalbumin, it is ovalbumin ? rabbit. Since the frequencies of the amide I band appear near 1655 cm^?1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.     

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.     

Purification and characterization of the serum ferroxidase inhibitor

The inhibitor of the serum ferroxidases, recently detected in rabbit serum, has been purified to homogeneity from human serum by a combination of gel-filtration and ion-exchange chromatography. The molecular weight, chromatographic behavior, electrophoretic mobility, electrofocusing pH, carbohydrate content, and reactivity with anti-human albumin during immunodiffusion indicate that the ferroxidase inhibitor is serum albumin. Copper-binding studies, proteolytic fragmentation studies, and a comparison of the inhibitory potencies of several albumin species which differ in their affinity for copper strongly indicate that albumin elicits its inhibitory effect on the serum ferroxidases by interacting with the functional copper of these enzymes. Kinetic analyses further suggest that albumin competes with substrate (ferrous iron) for binding to the functional copper of the serum ferroxidases.     

Polyclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II

Polyclonal antibodies against rabbit skeletal muscle phosphatases C-I and C-II were raised in goats and in mice. The goat polyclonal antibodies to phosphatases C-I and C-II were examined for their ability to immunoblot the purified enzymes and crude rabbit muscle extracts. In preparations of phosphatases C-I and C-II that were apparently homogeneous, the expected ca. 35- to 38-kDa polypeptides were immunoblotted, but, in addition, immunoblotting of a 67-kDa polypeptide was observed. Both the antisera blotted only the 67-kDa polypeptide in crude rabbit muscle extracts and not the expected 35- to 38-kDa polypeptides. These findings are qualitatively similar to those reported previously (D.L. Brautigan et al. (1985) J. Biol. Chem. 260, 4295-4305) where immunoblotting experiments with a sheep antisera to phosphatase C-I indicated that the ca. 35-kDa polypeptide originates from a 70-kDa precursor. On further investigation, it was found that our antisera were strongly immunoreactive to rabbit serum albumin. The antisera blotted purified rabbit albumin, but not bovine serum albumin. After passage through a rabbit albumin-Sepharose column, the antisera lost immunoreactivity to rabbit albumin, and no longer blotted the ca. 70-kDa band in muscle extracts or in purified enzyme preparations. These findings show that the phosphatase preparations contained traces of albumin which produced a strong antigenic reaction. Production of antisera in BALB/c mice produced similar results; i.e., an antibody to the low-molecular-weight phosphatases was produced that was also a strong antibody to rabbit albumin. This antibody could be removed by affinity adsoption on rabbit albumin-Sepharose columns. In addition, the antibodies to phosphatase C-I displayed no cross-reactivity to phosphatase C-II, while antibodies to C-II showed no cross-reactivity to phosphatase C-I by immunoblotting methods.     

Albumin binding as a general strategy for improving the pharmacokinetics of proteins

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules. Using peptide phage display, we identified a series of peptides having the core sequence DICLPRWGCLW that specifically bind serum albumin from multiple species with high affinity. These peptides bind to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites. Using surface plasmon resonance, the dissociation equilibrium constant of peptide SA21 (Ac-RLIEDICLPRWGCLWEDD-NH(2)) was determined to be 266 +/- 8, 320 +/- 22, and 467 +/- 47 nm for rat, rabbit, and human albumin, respectively. SA21 has an unusually long half-life of 2.3 h when injected by intravenous bolus into rabbits. A related sequence, fused to the anti-tissue factor Fab of D3H44 (Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., Schoenfeld, J., Lowe, D., Lai, J., Rancatore, P., Iverson, M., Lim, A., Chisholm, V., Kelley, R. F., Riederer, M., and Kirchhofer, D. (2001) Thromb. Haemost. 85, 379-389), enabled the Fab to bind albumin with similar affinity to that of SA21 while retaining the ability of the Fab to bind tissue factor. This interaction with albumin resulted in reduced in vivo clearance of 25- and 58-fold in mice and rabbits, respectively, when compared with the wild-type D3H44 Fab. The half-life was extended 37-fold to 32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving 25-43% of the albumin half-life in these animals. These half-lives exceed those of a Fab'(2) and are comparable with those seen for polyethylene glycol-conjugated Fab molecules, immunoadhesins, and albumin fusions, suggesting a novel and generic method for improving the pharmacokinetic properties of rapidly cleared proteins.     

Regulation of rabbit acute phase protein biosynthesis by monokines.

We defined the acute phase behaviour of a number of rabbit plasma proteins in studies (in vivo) and studied the effects of monokine preparations on their synthesis by rabbit primary hepatocyte cultures. Following turpentine injection, increased serum levels of C-reactive protein, serum amyloid A protein, haptoglobin, ceruloplasmin, and decreased concentrations of albumin were observed. In contrast to what is observed in man, concentrations of alpha 2-macroglobulin and transferrin were increased. Co-culture of primary hepatocyte cultures with lipopolysaccharide-activated human peripheral blood monocytes or incubation with conditioned medium prepared from lipopolysaccharide-activated human or rabbit monocytes resulted in dose-dependent induction of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and depression of albumin synthesis, while C-reactive protein synthesis and mRNA levels remained unchanged. A variety of interleukin-1 preparations induced dose-dependent increases in the synthesis and secretion of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and decreased albumin synthesis. Human recombinant tumour necrosis factor (cachectin) induced a dose-dependent increase in synthesis of haptoglobin and ceruloplasmin. In general, human interleukin-1 was more potent than mouse interleukin-1 and tumour necrosis factor. None of the monokines we studied had an effect on C-reactive protein synthesis or mRNA levels. These data confirm that C-reactive protein, serum amyloid A, haptoglobin and ceruloplasmin display acute phase behaviour in the rabbit, and demonstrate that, in contrast to their behaviour in man, alpha 2M and transferrin are positive acute phase proteins in this species. While both interleukin-1 and tumour necrosis factor regulate biosynthesis of a number of these acute phase proteins in rabbit primary hepatocyte cultures, neither of these monokines induced C-reactive protein synthesis. Comparison of these findings with those in human hepatoma cell lines, in which interleukin-1 does not induce serum amyloid A synthesis, suggests that the effect of interleukin-1 on serum amyloid A synthesis may be indirect.     

The binding of d-glucosyl-neoglycoproteins to the hepatic asialoglycoprotein receptor

The binding of D-glucosyl-neoglycoproteins and D-galactose-terminated glycoproteins to the hepatic asialoglycoprotein receptor of rabbit liver membranes were characterized and compared. The binding of both types of glycoproteins showed the same dependence on calcium concentration, sensitivity to neuraminidase, and degree of inhibition by various carbohydrate derivatives. These results, along with the observation that the rabbit liver membranes bound both the D-glucosyl- and D-galactosyl-terminated glycoproteins to the same extent, indicated that both types of glycoproteins bound to the same receptor. To confirm this hypothesis, receptors were isolated from rabbit livers by affinity chromatography using D-galactosyl-bovine serum albumin or D-glucosyl-bovine serum albumin immobilized on Sepharose. These receptors were shown to be identical by several chemical and immunological criteria as well as in their ability to bind equal amounts of D-galactosyl- and D-glucosyl-terminated glycoproteins. The conclusion is that the rabbit hepatic asialoglycoprotein receptor cannot discriminate between D-galactosyl and D-glucosyl-terminated glycoproteins and binds both.     

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

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.     

Identification of a "new" rabbit and human serum protein with fast electrophoretic mobility

A new protein has been identified in both rabbit and human serum. The salient characteristic of this protein is its high negative charge as revealed by its rapid anodal migration during electrophoresis at alkaline pH. This protein has tentatively been designated fast-moving protein because of its electrophoretic mobility. Molecular weight determination by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the molecular weight was 85,000 daltons. A goat antiserum made to the rabbit fast-moving protein cross-reacted with both rabbit and human serum albumin. Although no apparent structural relationship between fast-moving protein and albumin was found by peptide-mapping studies, a peptide with a molecular weight of 24,000 daltons and with antigenic determinants in common with rabbit fast-moving protein, was isolated from cyanogen bromide-treated human serum albumin. The structural relationship between fast-moving protein and albumin is discussed.     

Selection of affinity ligands for protein purification from proteolytic digests

A simple method for the selection of affinity ligands from proteolytic digests by affinity chromatography is proposed. A small proportion of the peptides in the trypsin digest of bovine serum albumin (BSA) or the pepsin digest of cytochrome are retarded on insulin-immobilised or HSA (human serum albumin)-immobilised affinity columns, respectively. The peptides in these selected fractions can be immobilised onto solid phases and used in affinity chromatography procedures for the purification of insulin or HSA. © Rapid Science Ltd. 1998     

Only the initial binding of cationic immune complexes to glomerular anionic sites is mediated by charge-charge interactions

The role of charge-charge interactions between cationic immune complexes and the anionic sites on the glomerular basement membrane was examined. For this purpose, soluble immune complexes at fivefold antigen excess were prepared with human serum albumin and cationized rabbit antibodies to this protein. When unrelated cationic proteins, protamine sulfate or cationized rabbit serum albumin, were given 1 min before the cationized immune complexes, glomerular immune deposits did not form. Cationic immune complexes allowed to deposit in glomeruli could readily be displaced by protamine sulfate or cationized rabbit serum albumin injected 1 min after the immune complexes. If the same cationic molecules were injected 1 hr after the immune complexes, the complexes could not be displaced from glomeruli. In contrast, cationic complexes that were deposited in glomeruli in the presence of a very high degree of antigen excess in circulation to prevent their condensation into larger complexes in glomeruli were readily displaced at 1 min and 1 hr with protamine sulfate or with cationized rabbit serum albumin. On the basis of these results, we concluded that the initial binding of cationic immune complexes to glomeruli occurs by charge-charge interactions. Once the immune complexes in glomeruli condense to larger deposits, forces other than charge-charge interactions are responsible for their retention in glomeruli.     

Rabbit liver growth hormone receptor and serum binding protein. Purification, characterization, and sequence

A putative growth hormone receptor from detergent-solubilized rabbit liver membranes and the growth hormone binding protein from rabbit serum have been purified 59,000- and 400,000-fold, respectively, primarily by affinity chromatography. Both purified proteins exhibit high affinity binding for human growth hormone; K alpha = 9-30 x 10(9) M-1 for the liver receptor and K alpha = 6 x 10(9) M-1 for the binding protein. The apparent molecular weight of the liver receptor is 130,000 by reduced sodium dodecyl sulfate gel electrophoresis, while that of the binding protein is 51,000. Both contain N-linked carbohydrate. The amino-terminal sequences of the liver growth hormone receptor and the serum binding protein were found to be the same, indicating that the binding protein corresponds to the extracellular domain of the liver receptor. Ubiquitin was found covalently linked to the liver receptor but not to the serum binding protein. The amino acid sequences of several peptides from the liver receptor were also determined after tryptic and V8 protease digestion.     

The effect of ligand presaturation on the interaction of serum albumins with an immobilized Cibacron Blue 3G-A studied by affinity gel electrophoresis.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.     

A factor that activates oscillatory chloride currents in Xenopus oocytes copurifies with a subfraction of serum albumin

Vertebrate blood sera contain a factor that elicits oscillatory chloride currents in Xenopus oocytes through activation of the phosphatidylinositol second messenger system. This factor was purified from rabbit and human sera by a sequence of Blue-Agarose chromatography, concanavalin A affinity chromatography, and hydroxyapatite fractionation, yielding a single active protein band (67 kDa). This protein is a subfraction of serum albumin, as revealed by its molecular mass, isoelectric properties, peptide maps, amino acid composition, and NH2-terminal sequence. Moreover, the factor could be purified with a monoclonal antibody to serum albumin and its ability to elicit oscillatory currents was inhibited by several polyclonal-monospecific antibodies to serum albumin. Various commercial high purity albumin preparations elicited oscillatory currents in oocytes. The activity of albumin was partially reduced by charcoal absorption and was greatly diminished when crystalline albumin was extracted with dry methanol. However, the activity was resistant to extraction with chloroform/ether, disulfide cleavage, and denaturation with 8 M urea, 6 M guanidinium chloride, and 1% sodium dodecyl sulfate. Trypsin or lipase treatment substantially reduced the potency of the active albumin, but neither treatment alone abolished the factor even after prolonged digestion. In contrast to serum or serum albumin, freshly collected blood plasma or purified plasma albumin did not evoke oscillatory currents. This indicates that some of the plasma albumin changes during blood coagulation and acquires a "factor" that makes it capable of activating the phosphatidylinositol-Ca2+ system in Xenopus oocytes. The serum factor also activates the phosphatidylinositol system in a variety of mammalian cells, suggesting that the modified albumin may play a role in cellular events related to tissue repair following injury.     

Mass spectrometric identification of serum peptides employing derivatized poly(glycidyl methacrylate/divinyl benzene) particles and mu-HPLC

Biomarkers play a key role in preclinical screening and diagnosis of a disease. Various support materials are utilized for this task, in combination with MALDI-TOF-MS. The way to effectively bind serum contents and their profiling is well-elaborated by the material-enhanced laser desorption ionization (MELDI) approach. In this particular work, focus is placed on the development of a strategy to identify low molecular weight serum peptides. Poly(GMA/DVB) is derivatized in a way to achieve an affinity termed as immobilized metal ion affinity chromatography (IMAC). Iminodiacetic acid (IDA) is used as a chelating ligand, whereas copper (Cu2+) acts as a metal ion for complexing peptides and proteins out of blood serum. Polymer binds the serum compounds over a broad mass range, which includes low mass peptides and high mass albumin (66 kDa). Bound contents are eluted from material by an acetonitrile/trifluoroacetic acid mixture, which proves the reversible nature of metal and amino acid linkage. Polystyrene/divinyl benzene (PS/DVB) monolithic capillary column is used for fractionation through RP-HPLC, prior to the target spotting. The tandem TOF fragment ion mass spectra of each fraction is acquired and used to search against the Swiss-Prot database, using the Mascot search engine for the identification of peptides.