Recombinant Human Serum Albumin Research Articles

Molecular Capture in Protein Nanotubes

We describe molecular capturing properties of protein nanotubes with a controllable ligand binding affinity and size selectivity. These practical biocylinders were prepared using an alternating layer-by-layer (LbL) assembly of protein and oppositely charged poly(amino acid) into the nanoporous polycarbonate (PC) membrane (pore diameter, 400 nm), with subsequent dissolution of the template. The tube wall typically comprises six layers of poly-L-arginine (PLA) and human serum albumin (HSA) [(PLA/HSA)(3)]. Use of high molecular weight PLA (M(w) = ca. 70 000) yielded robust nanotubes, which are available as lyophilized powder. The (PLA/HSA)(3) nanotubes swelled considerably in water, although the outer diameter was almost unaltered. Uranyl ion, 3,3'-diethylthiacarbocyanine iodide, and zinc(II) protoporphyrin IX (ZnPP) were bound to the HSA component in the cylinder wall. Similar nanotubes comprising recombinant HSA mutant [rHSA(His)], which has a strong binding affinity for ZnPP, captured this ligand more tightly. Furthermore, addition of excess myristic acid released ZnPP from the tubes through a ligand replacement reaction. The hybrid nanotubes bearing a single avidin layer as an internal surface captured FITC-biotin efficiently. Biotin-labeled nanoparticles are also incorporated into the tubes when their particle size is sufficiently small to enter the pores. Subsequent TEM observation revealed a line of loaded nanoparticles (100 nm) in the one-dimensional space interior.

FcRn binding properties of an abnormal truncated analbuminemic albumin variant

Background: The major histocompatibility class I-related neonatal Fc receptor, FcRn, salvages both IgG and albumin from degradation and thus contributes to maintain high serum levels of these proteins. Analbuminemia is a rare autosomal recessive disorder characterized by clinically observed allelic albumin variants that are absent or found in very low concentrations in the blood circulation. Such variants may have altered FcRn binding properties that affect their half-life, biodistribution and thereby transport ability. Methods: We established an easy cloning, expression and purification strategy to obtain recombinant GST-tagged human serum albumin (HSA) variants for evaluation of pH dependent FcRn binding properties using an enzyme-linked immunosorbent assay (ELISA) and a real time surface plasmon resonance (SPR) biosensor system. Results: The strategy yielded purified GST-tagged albumin variants. A recombinant truncated HSA variant similar to a clinically observed splice mutant denoted Bartin, here abrogated HSA Bartin, showed no detectable pH dependent FcRn binding compared to a fully functional albumin wild type variant, HSA Wt, and a truncated HSA variant consisting of only the carboxy terminal domain III (HSA DIII). Conclusions: The approach described can be used to rapidly screen clinically observed truncated or otherwise mutant or modified HSA variants regarding their pH dependent FcRn binding properties. Here, we demonstrate that a recombinant truncated HSA variant, HSA Bartin, does not interact with FcRn, which gives a molecular explanation for the low serum levels. In addition, DIII of HSA alone was shown to retain its FcRn binding property.

A Transient Inflammatory Reaction in the Lung After Experimental Hemorrhagic Shock and Resuscitation With a Hemoglobin-Vesicles Solution Compared With Rat RBC Transfusion

Transfusion for hemorrhagic shock can improve oxygenation, but immunoreactions may induce inflammation. Artificial oxygen carriers have been developed to address clinical concerns of infection and stability, but whether an artificial oxygen carrier might induce inflammation is not well known. To address this question, we compared inflammatory reactions after resuscitation with hemoglobin vesicles (HbVs) or red blood cells (RBCs) in a hemorrhagic shock rat model. Both HbVs and the stored and irradiated rat RBCs deprived of buffy coat were suspended in recombinant human serum albumin [(Hb) = 8.6 g/dL]. Under anesthesia, hemorrhagic shock was induced for 30 min, followed by resuscitation by 20 min transfusion of HbVs or rat RBCs in a volume equivalent to the volume of withdrawn blood. Lungs were excised 2 or 24 h after resuscitation, and mRNA levels of tumor necrosis factor alpha (TNF-alpha), intercellular adhesion molecule-1 (ICAM-1), nitric oxide synthase 2 (iNOS), nitric oxide synthase 3, hypoxia-inducible factor 1 alpha, and heme oxygenase 1 (HO-1) were measured. In rats resuscitated with HbVs, mRNA levels of TNF-alpha and HO-1 2 h after resuscitation were significantly higher than those in the rat RBC group, but the levels at 24 h were similar in both groups. The expression of iNOS and ICAM-1, second messengers of inflammation, was not affected, and inflammatory levels after 24 h with HbVs are similar to rat RBC transfusion. The rat RBC group did not show an expected inflammatory reaction related to a transfusion-induced lung injury, and a clinical relevance concerning this level of transient inflammatory reaction induced by HbVs is not known; however, attention to the early stage of resuscitation in ongoing studies of HbV is required.

O2 Binding Properties of Human Serum Albumin Quadruple Mutant Complexed Iron Protoporphyrin IX with Axial His-186 Coordination

Abstract The O2 binding properties of complexes of iron(II) protoporphyrin IX with quadruple mutants of recombinant human serum albumin (rHSA) that provide axial His-186 coordination have been characterized; their O2 binding parameters were similar to those of analogues having proximal His-185 and of human red blood cells.

Quantification of Urinary Albumin by Using Protein Cleavage and LC-MS/MS

Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, (15)N-labeled recombinant HSA ((15)NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides (42)LVNEVTEFAK(51) and (526)QTALVELVK(534) from albumin and (15)NrHSA were referenced to the calibration curve. The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13-200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%-11.0% for the peptide (42)LVNEVTEFAK(51), and 1.9%-12.3% for the (526)QTALVELVK(534) peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%-14.8% for the (42)LVNEVTEFAK(51) peptide, and 6.4%-14.1% for the (526)QTALVELVK(534) peptide. For the (42)LVNEVTEFAK(51) peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R(2) value of 0.97 and a regression line of y = 0.99x + 23.16. Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a (15)NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.

Use of recombinant human serum albumin in pediatric patients with nephrotic syndrome

Sirs, We have read with great interest the article of Dharmaraj et al. [1]. In a clinical setting, some patients with nephrotic syndrome (NS) who are unresponsive to diuretic therapy alone develop severe edema, pleural effusion and ascites. In this context, the administration of human serum albumin in combination with the infusion of loop diuretics is the treatment of choice, although this treatment regimen remains controversial. Since plasma-derived human serum albumin (pHSA) is manufactured from human blood, the supply is limited and the risk of infection by unknown viruses or prions cannot be completely excluded [2]. Recombinant human serum albumin (rHSA) has recently been developed from Pichia pastoris without the use of any animal-derived materials [3], and the clinical use of 25% rHSA for patients suffering from significant albumin loss due to burns, NS and liver cirrhosis, among other conditions, has been approved by the Ministry of Health and Welfare in Japan. Although rHSA has been successfully applied without significant serious adverse effects in the treatment of adult patients with hemorrhagic shock, NS, burns, acute abdominal complaints and liver cirrhosis, poorrisk patients before surgery and post-open heart surgery patients [3–5], little information is currently available on the use of rHSA for the treatment of pediatric patients with NS. Here, we report our preliminary experience of using of rHSA in pediatric patients with NS. Seven patients with NS [four patients with minimal change NS (MCNS), two with focal segmental glomerulosclerosis (FSGS) and one with Henoch–Schonlein purpura nephritis (HSPN)] were initially examined in our study. We started rHSA treatment after receiving both the approval of the ethics committee of our institution and written informed consent from each of the patients and their parents. Specific immunoglobulin (Ig)E antibody titers against Pichia yeast components were measured before the start of rHSA administration. If the serum IgE antibody was positive for Pichia yeast, rHSA administration was prohibited. Of the seven patients initially included in the study, one patient with MCNS who showed weak positivity for serum specific IgE antibody was excluded. The remaining six patients received rHSA treatment in combination with the infusion of loop diuretics for the treatment of refractory edema at Hirosaki University Hospital between September 2008 and January 2009. Among these six patients, one patient with FSGS received three separate rHSA treatments, with the treatment repeated every other month. Thus, a total of eight clinical observations were evaluated. Intravenous rHSA (25% rHSA; Medway, Bipha Co, Chitose, Japan) was administered at a dose of 0.3–0.5 g/kg, one to three times daily in combination with furosemide (1 mg/kg). One treatment course of rHSA was scheduled for at least three consecutive days of administration. Since this study was initially designed mainly to evaluate the safety of rHSA for the treatment of childhood NS, the treatment protocol was individualized according to each patient’s status. Allergic adverse drug reactions to rHSA were carefully monitored during the study period. After 1 month of treatment, a repeat measurement of serum specific IgE antibody against Pichia yeast components was performed, revealing that serum specific IgE antibody for Pichia had remained Pediatr Nephrol (2009) 24:2275–2276 DOI 10.1007/s00467-009-1221-y

Static Structures and Dynamics of Hemoglobin Vesicle (HbV) Developed as a Transfusion Alternative

Hemoglobin vesicle (HbV) is an artificial oxygen carrier that encapsulates solution of purified and highly concentrated (ca. 38 g dL(-1)) human hemoglobin. Its exceptionally high concentration as a liposomal product (ca. 40% volume fraction) achieves an oxygen-carrying capacity comparable to that of blood. We use small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) to investigate the hierarchical structures and dynamics of HbVs in concentrated suspensions. SAXS data revealed unilamellar shell structure and internal density profile of the artificial cell membrane for Hb encapsulation. The SAXS intensity of HbV at scattering vector q > 0.5 nm(-1) manifests dissolution states of the encapsulated Hbs in the inner aqueous phase of the vesicle having ca. 240 nm diameter. The peak position as well as the height and width of static structure factor of Hb before and after encapsulation are almost identical, demonstrating the preserved protein-protein interactions in the confined space. To overcome multiple scattering from turbid samples, we employed thin layer-cell DLS combined with the so-called bruteforce and echo techniques, which allows us to observe collective diffusion dynamics of HbVs without dilution. A pronounced slowdown of the HbV diffusion and eventual emergence of dynamically arrested state in the presence of high-concentration plasma substitutes (water-soluble polymers), such as dextran, modified fluid gelatin, and hydroxylethyl starch, can be explained by depletion interaction. A significantly weaker effect of recombinant human serum albumin on HbV flocculation and viscosity enhancement than those induced by other polymers is clearly attributed to the specificity as a protein; its compact structure efficiently reduces the reservoir polymer volume fraction that determines the depth of the attractive potential between HbVs. These phenomena are technically essential for controlling the suspension rheology, which is advantageous for versatile clinical applications.

Purification of fusion protein HSA/IL1ra and its bioactivity

To purify the recombinant human serum albumin fusion protein with interleukin 1 (HAS/IL1ra) and to detect the bioactivity of the fusion protein. The recombinant HAS/IL1ra protein was purified by affinity chromatography and ion exchange chromatography, the bioactivity of the fusion protein was detected by IL1-induced A375 S2 cell killing. The purity of the fusion protein was at least 98 % as assessed by HPLC and the protective effect from the IL1-induced A375 S2 cell killing was similar to natural IL1ra. The purified recombinant HAS/IL1ra protein in this study has a satisfactory bioactivity.

Allosteric and binding properties of Asp1–Glu382 truncated recombinant human serum albumin – an optical and NMR spectroscopic investigation

Human serum albumin (HSA) is known for its exceptional ligand-binding capacity; indeed, its modular domain organization provides a variety of ligand-binding sites. Its flexible modular structure involves more than the immediate vicinity of the binding site(s), affecting the ligand-binding properties of the whole protein. Here, biochemical characterization by (1)H-NMR relaxometry and optical spectroscopy of a truncated form of HSA (tHSA) encompassing domains I and II (Asp1-Glu382) is reported. Removal of the C-terminal domain III results in a number of contacts that involve domain I (containing the heme site) and domain II (containing the warfarin site) being lost; however, the allosteric linkage between heme and warfarin sites is maintained. tHSA shows a nuclear magnetic relaxation dispersion profile similar to that of HSA, and displays increased affinity for ibuprofen, warfarin, and heme, suggesting that the fold is preserved. Moreover, the allosteric properties that make HSA a peculiar monomeric protein and account for the regulation of ligand-binding modes by heterotropic interactions are maintained after removal of domain III. Therefore, tHSA is a valuable model with which to investigate allosteric properties of HSA, allowing independent analysis of the linkages between different drug-binding sites.

Expanding the Genetic Repertoire of the Methylotrophic Yeast Pichia pastoris

To increase the utility of protein mutagenesis with unnatural amino acids, a recombinant expression system in the methylotrophic yeast Pichia pastoris was developed. Aminoacyl-tRNA synthetase/suppressor tRNA (aaRS/tRNA(CUA)) pairs previously evolved in Saccharomyces cerevisiae to be specific for unnatural amino acids were inserted between eukaryotic transcriptional control elements and stably incorporated into the P. pastoris genome. Both the Escherichia coli tyrosyl- and leucyl-RS/tRNA(CUA) pairs were shown to be orthogonal in P. pastoris and used to incorporate eight unnatural amino acids in response to an amber codon with high yields and fidelities. In one example, we show that a recombinant human serum albumin mutant containing a keto amino acid (p-acetylphenylalanine) can be efficiently expressed in this system and selectively conjugated via oxime ligation to a therapeutic peptide mimetic containing an permittivity-(2-(aminooxy)acetyl)-L-lysine residue. Moreover, unnatural amino acid expression in the methylotrophic host was systematically optimized by modulation of aaRS levels to express mutant human serum albumin in excess of 150 mg/L in shake flasks, more than an order of magnitude better than that reported in S. cerevisiae. This methodology should allow the production of high yields of complex proteins containing unnatural amino acids whose expression is not practical in existing systems.

Immunogencity of HSA-L7/L12 (Brucella abortus ribosomal protein) in an animal model.

The immunogenic Brucella abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis. This study was aimed to evaluate the protection of recombinant Human Serum Albumin (HAS)-L7/L12 fusion protein in Balb/c mice. The amplified L7/L12 gene was cloned in pYHSA5 vector, pYHSA5-L7/L12 construct was transformed in Saccharomyces cerevisiae and the expressed protein from supernatant was purified by affinity chromatography. Balb/c mice were immunized in five groups by tHSA-L7/L12 fusion protein (group 1), Brucella abortus S19 (group 2), HSA (group 3), recombinant L7/L12 (group 4), PBS (group 5). ELISA to detect antibody production, LTT test to assess antigen specific lymphocyte response were conducted prior to virulent B. abortus strain 544 challenge two weeks after the last injection. Bacterial counts from spleens of immunized mice were done four weeks after challenge. In ELISA tests, the specific antibodies exhibited a dominance of immunoglobulin IgG1 over IgG2a. In addition, the tHSA-L7/L12 fusion protein and L7/L12 elicited a strong T-cell proliferative response upon restimulation in vitro with recombinant tHSA-L7/L12 and L7/L12, suggesting the induction of a cellular immunity response in vivo. However, there was no significant difference in proliferative response of L7/L12 and tHSA-L7/L12 fusion protein (p>0.05). The L7/L12 and tHSA-L7/L12 fusion protein vaccines could also induce significant protection against challenge with the virulent strain B. abortus 544 in Balb/c mice (p< or =0.05). The tHSA-L7/L12 fusion protein, similar to L7/L12 has the ability to induce antigen specific lymphocyte proliferation, stimulate humoral immunity and engender protection.

Artificial Oxygen Carriers, Hemoglobin Vesicles and Albumin−Hemes, Based on Bioconjugate Chemistry

Hemoglobin (Hb, Mw: 64 500) and albumin (Mw: 66 500) are major protein components in our circulatory system. On the basis of bioconjugate chemistry of these proteins, we have synthesized artificial O(2) carriers of two types, which will be useful as transfusion alternatives in clinical situations. Along with sufficient O(2) transporting capability, they show no pathogen, no blood type antigen, biocompatibility, stability, capability for long-term storage, and prompt degradation in vivo. Herein, we present the latest results from our research on these artificial O(2) carriers, Hb-vesicles (HbV) and albumin-hemes. (i) HbV is a cellular type Hb-based O(2) carrier. Phospholipid vesicles (liposomes, 250 nm diameter) encapsulate highly purified and concentrated human Hb (35 g/dL) to mimic the red blood cell (RBC) structure and eliminate side effects of molecular Hb such as vasoconstriction. The particle surface is modified with PEG-conjugated phospholipids, thereby improving blood compatibility and dispersion stability. Manipulation of physicochemical parameters of HbV, such as O(2) binding affinity and suspension rheology, supports the use of HbV for versatile medical applications. (ii) Human serum albumin (HSA) incorporates synthetic Fe(2+)porphyrin (FeP) to yield unique albumin-based O(2) carriers. Changing the chemical structure of incorporated FeP controls O(2) binding parameters. In fact, PEG-modified HSA-FeP showed good blood compatibility and O(2) transport in vivo. Furthermore, the genetically engineered heme pocket in HSA can confer O(2) binding ability to the incorporated natural Fe(2+)protoporphyrin IX (heme). The O(2) binding affinity of the recombinant HSA (rHSA)-heme is adjusted to a similar value to that of RBC through optimization of the amino acid residues around the coordinated O(2).

FLUID RESUSCITATION WITH ARTIFICIAL OXYGEN CARRIERS IN HEMORRHAGED RATS

Polyethylene glycol (PEG)-modified hemoglobin (Hb) vesicles (HbVs) are artificial oxygen carriers encapsulating a concentrated Hb solution in phospholipid vesicles. In our previous studies, HbV showed a sufficient resuscitative effect comparable to that of red blood cells in hemorrhagic shock animal models during several hours' observation. However, the profiles of the recovery, including hematopoiesis and elimination of HbV, remain unknown. This study conducted 14-day observations of Wistar rats after hemorrhagic shock and fluid resuscitation with HbV suspended in recombinant human serum albumin. Shock was induced by 50% blood withdrawal from a femoral artery. The rats showed hypotension, metabolic acidosis, and hyperventilation. After 15 min, they received HbV or shed autologous blood through a femoral vein. Both groups showed rapid recovery of hemodynamic and blood gas parameters. No meaningful difference was found between groups. After decannulation and awakening, the rats were housed in cages. The reduced hematocrit of the HbV group returned to the original level in 7 days. Plasma enzyme levels were slightly higher in both groups at 1 day because of systemic reperfusion injury. Splenomegaly was considerable in the HbV group because of the HbV accumulation and extramedullar hematopoiesis, but it subsided within 14 days. Along with the HbV elimination in the spleen and liver, immunohistochemistry with anti-PEG antibody revealed that PEG-conjugated lipid had disappeared within 14 days. In conclusion, HbV showed a sufficient resuscitative effect comparable to that of red blood cell transfusion. Phagocytized HbV disappeared within 14 days. Elevated hematopoiesis contributed to complete hematocrit recovery within 7 days.

The role of an amino acid triad at the entrance of the heme pocket in human serum albumin for O2 and CO binding to iron protoporphyrin IX

Complexation of iron(II) protoporphyrin IX (Fe(2+)PP) into a genetically engineered heme pocket on recombinant human serum albumin (rHSA) creates an artificial hemoprotein which can bind O(2) reversibly at room temperature. Here we highlight a crucial role of a basic amino acid triad the entrance of the heme pocket in rHSA (Arg-114, His-146, Lys-190) for O(2) and CO binding to the prosthetic Fe(2+)PP group. Replacing His-146 and/or Lys-190 with Arg resolved the structured heterogeneity of the possible two complexing modes of the porphyrin and afforded a single O(2) and CO binding affinity. Resonance Raman spectra show only one geometry of the axial His coordination to the central ferrous ion of the Fe(2+)PP.

Addition of a sequence from α2-antiplasmin transforms human serum albumin into a blood clot component that speeds clot lysis

BackgroundThe plasma protein α2-antiplasmin (α2AP) is cross-linked to fibrin in blood clots by the transglutaminase factor XIIIa, and in that location retards clot lysis. Competition for this effect could be clinically useful in patients with thrombosis. We hypothesized that fusion of N-terminal portions of α2-antiplasmin to human serum albumin (HSA) and production of the chimeric proteins in Pichia pastoris yeast would produce a stable and effective competitor protein.ResultsFusion protein α2AP(13-42)-HSA was efficiently secreted from transformed yeast and purified preparations contained within a mixed population the full-length intact form, while fusions with longer α2AP moieties were inefficiently secreted and/or degraded. The α2AP(13-42)-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native α2AP. Excess α2AP(13-42)-HSA competed with α2AP for cross-linking to chemical lysine donors more effectively than a synthetic α2AP(13-42) peptide, and reduced the α2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide. Native α2AP was found in in vivo clots in rabbits to a greater extent than α2AP(13-42), however.ConclusionIn this first report of transfer of transglutamination substrate status from one plasma protein to another, fusion protein α2AP(13-42)-HSA was shown to satisfy initial requirements for a long-lasting, well-tolerated competitive inhibitor of α2-antiplasmin predicted to act in a clot-localized manner.

小児ネフローゼ症候群患者に対する遺伝子組換え人血清アルブミン製剤の使用経験

小児ネフローゼ症候群患者に対する遺伝子組換え人血清アルブミン製剤の使用経験

A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo

The leech protein hirudin is a potent inhibitor of thrombin, but clinical use of recombinant hirudin is restricted by haemorrhagic risks, and complicated by hirudin's rapid clearance from the circulation. We previously employed albumin fusion to slow hirudin variant 3 (HV3) clearance. In this study, we hypothesized that reconfiguration of the chimera, appending human serum albumin (HSA) to the N-terminus of HV3, with an intervening plasmin cleavage site, would create a slowly cleared, plasmin-activatable HV3. Potential plasmin cleavage sites were screened by expression in Escherichia coli, interposed between glutathione sulfotransferase and HV3 domains. The most reactive sequence (GSGIYR-ITY) was recreated in C-terminally His-tagged albumin fusion protein HSACHV3, expressed in Pichia pastoris yeast and purified by nickel-chelate affinity chromatography. HSACHV3 showed no thrombin inhibitory activity in the absence of plasmin, but liberated active HV3 in a time- and concentration-dependent manner in its presence. In a discontinuous clot assay involving clot-bound thrombin, HSACHV3 assisted clot lysis by limiting clot extension in a tPA- and concentration-dependent manner. Similar results were obtained in plasma at higher concentrations of HSACHV3. The chimeric protein exhibited much slower clearance in mice than unfused HV3, and indistinguishable pharmacokinetics from unfused recombinant HSA. In a mouse tail transection bleeding model, doses of HSACHV3 identical to those of HV3 that elicited a four-fold increase in the volume of shed blood were without effect. Our results suggest that HSACHV3 is a fully latent, plasmin activatable, long-lasting hirudin, of potential benefit in thrombotic disorders resistant to natural or pharmacological clot lysis.

Synthesis and characterization of recombinant factor VIIa‐conjugated magnetic iron oxide nanoparticles for hemophilia treatment

Maghemite (gamma-Fe(2)O(3)) nanoparticles of 15.0 +/- 2.1 nm in diameter were prepared by nucleation, followed by controlled growth of magnetic iron oxide thin films onto gelatin nuclei. Functionalization of these magnetic nanoparticles with activated double bonds was accomplished by interacting divinyl sulfone with the gelatin coating of the gamma-Fe(2)O(3) nanoparticles. The activated double bonds were then used for covalent binding, via Michael addition reaction, of recombinant factor VIIa and human serum albumin to the surface of these nanoparticles. Recombinant factor VIIa was also physically bound to the magnetic nanoparticles by interacting this factor with the human serum albumin conjugated gamma-Fe(2)O(3) nanoparticles. The influence of factor VIIa concentration on the immobilization yield has been elucidated. Leakage of the bound factor VIIa into PBS containing 4% albumin was insignificant. The coagulant activity of the physically adsorbed recombinant factor VIIa was similar to that of the free one and was significantly better than that of the covalently bound. The blood half-life of free factor VIIa is short, about 2-3 h, because of digestion by proteolytic enzymes and inhibitory effects. Stabilization of factor VIIa against trypsin (a model proteolytic enzyme) and chloromethyl ketone-type inhibitor was accomplished by conjugation of the factor to the gamma-Fe(2)O(3) nanoparticles. This stabilization may extend the blood half-life of factor VIIa. Therefore, IV injection of factor VIIa conjugated gamma-Fe(2)O(3) nanoparticles instead of free factor may avoid the frequent dosing and reduce the cost of hemophilia treatment.

Preparation of S-sulfo albumin film and its cell adhesive property

Recently, large-scale production of the pharmaceutical grade recombinant human serum albumin was achieved, and several clinical trials have proved its safety and efficacy. Albumin is thought to be a candidate for a safe biopolymer sources for application to biomaterials. In this study, we treated albumin with sodium sulfite and sodium tetrathionate to give S-sulfo albumin, which was found to loose native albumin structure by CD spectra analysis and dye-binding assay. A water-insoluble S-sulfo albumin films were prepared by drying S-sulfo albumin solution and subsequent reformation of disulfide bonds by the oxidation with iodine. Ultimate strength, ultimate elongation and Young's modulus of S-sulfo albumin film prepared at room temperature were 3.3 ± 0.4 MPa, 30.8 ± 3.2% and 40.8 ± 3.3 MPa before oxidative treatment and changed to 13.8 ± 4.2 MPa, 5.6 ± 2.8% and 401.7 ± 15.3 MPa after oxidative treatment. When the film was prepared at 60 °C, similar tendency was observed. Thus, the disulfide bonds formation between albumin molecules by oxidative treatment converted the film stronger and stiffer. Cell adhesion and proliferation on the films were evaluated using mouse L929 fibroblast cells. Cell adhesion largely depended on the albumin structure; that is, cells did not attach to native albumin coated surfaces, while cell adhesion and proliferation occurred on the S-sulfo albumin films which lost their native albumin structure. Eighty percent of seeded cells were adhered on S-sulfo albumin films and proliferated well in a similar manner to those on the conventional culture dish. Our results indicate that S-sulfo albumin is a favorable cell culture substrate.

Purification of Recombinant Human Serum Albumin (rHSA) Produced by Genetically Modified Pichia Pastoris

Recombinant human serum albumin (rHSA) was produced by genetically transformed Pichia pastoris yeast. The cell-culture supernatant (CCS) contained 8–12 g/l rHSA that was purified in a three-step procedure involving (1) a capture step using the newly developed cation exchanger CaptoTM MMC; (2) an intermediate step using Phenyl SepharoseTM and, (3) a polishing step using Aminobutyl SepharoseTM 6 FF. The total recovery was 25–35% and the product fulfils the purity criteria of the European Pharmacopeia. Purified rHSA and plasma-derived HSA were essentially identical judging by SDS- or native-PAGE, and the pigment level (expressed as A350/A280) in the rHSA was 0.03 or less and was strongly dependent on the quality of the CCS. Dimers and polymers in the final product were less than that found in purified plasma-derived HSA. The molar mass of the purified rHSA, as well as of its natural counterpart, is 67 000 Daltons by MALDI-ToF mass spectrometry, while the iso-electric points of both recombinant and natural HSA ranged between pH 5.42–5.55 when determined in 8 M urea. The stability profiles of both proteins after heat treatment were identical as determined by differential scanning calorimetry (DSC). The results obtained here suggest the purified rHSA to be a homogeneous protein identical to its natural counterpart.