Human Serum Albumin Fusion Protein Research Articles

Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

BackgroundThe transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α2-antiplasmin (α2AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α2AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α2AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.ResultsExpression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H6NQEQVSPLTLLAG4Y (designated XL1); H6DQMMLPWAVTLG4Y (XL2); H6WQHKIDLPYNGAG4Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α2AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α2AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.ConclusionsFusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into in vivo clots formed in thrombosis models in both mice and rabbits.

Expression and characterization of recombinant human serum albumin fusion protein with C-peptide

C-peptide (CP), connecting the A and B chains in proinsulin, has been considered to possess physiological effects in diabetes. In order to prolong the half-life of CP in vivo, a long acting CP analog [human serum albumin (HSA-CP)] was obtained by direct gene fusion of a single-chain CP to HSA and expressed in host Pichia pastoris GS115. After 72 h of growth on methanol, the recombinant HSA-CP concentration reached a level of 145 mg/L, representing 70% of total proteins in culture supernatant. The recombinant fusion protein was purified by ultra filtration, Q-sepharose fast flow column and Superdex 75 size-exclusion column. It was specifically recognized by the anti-human HSA antibody and CP antibody in Western blotting assay. In vitro, the recombinant HSA-CP can stimulate HEK293 cell proliferation. In Zucker diabetic fatty (ZDF) rats, 12 weeks recombinant HSA-CP treatment could prevent the accumulation of glomerular extracellular matrix, which leads to mesangial expansion and glomerular hypertrophy. The terminal biological half-time of the protein was 3.38±1.87 h after single administration of 500 nmol/kg of HSA-CP in Wister rats. The pharmacokinetic analysis of the protein indicate its promising application in clinical medicine. Key words : C-peptide, human serum albumin, recombinant fusion protein, Pichia pastoris, bioactivity,biological half-time.

Fusion of HSA influences TNF-α neutralizing activity of shTNFRs

Soluble human tumor necrosis factor receptors (shTNFRI and shTNFRII) are antagonists of tumor necrosis factor-α (TNF-α) and are under clinical investigation as therapy for autoimmune diseases and transplant rejection. However, shTNFRI and shTNFRII are limited for clinical usage because of their short half-lives in vivo. Recombinant TNF-α receptors (infliximab and etanercept) are used in treatment of rheumatoid arthritis and Crohn's disease but are also being tested for a number of other autoimmune diseases. Human serum albumin (HSA) has been used to construct long-acting fusion proteins. Here, we report the effect of fusion of HSA with shTNFRI and with shTNFRII on shTNFR's neutralizing activity against TNF-α. HSA fusion proteins were separately expressed in Pichia pastoris. Purified recombinant shTNFRI-HSA, HSA-shTNFRI and HSA-shTNFRII could block the cytolytic activity of TNF-α in L929 cells, and the fusion at N-terminus of shTNFRI could result in larger degree of activity decline than that at the C-terminus. Activity of three fusion proteins was much weaker than etanercept, which demonstrated that fusion of HSA significantly influenced TNF-α neutralizing activity of shTNFRs. Compared with Fc fragment, HSA fusion technology may therefore not be an ideal strategy in development of long-acting shTNFRs protein drugs.

Expression and characterization of recombinant human serum albumin fusion protein with C-peptide

C-peptide (CP), connecting the A and B chains in proinsulin, has been considered to possess physiological effects in diabetes. In order to prolong the half-life of CP in vivo, a long acting CP analog [human serum albumin (HSA-CP)] was obtained by direct gene fusion of a single-chain CP to HSA and expressed in host Pichia pastoris GS115. After 72 h of growth on methanol, the recombinant HSA-CP concentration reached a level of 145 mg/L, representing 70% of total proteins in culture supernatant. The recombinant fusion protein was purified by ultra filtration, Q-sepharose fast flow column and Superdex 75 size-exclusion column. It was specifically recognized by the anti-human HSA antibody and CP antibody in Western blotting assay. In vitro, the recombinant HSA-CP can stimulate HEK293 cell proliferation. In Zucker diabetic fatty (ZDF) rats, 12 weeks recombinant HSA-CP treatment could prevent the accumulation of glomerular extracellular matrix, which leads to mesangial expansion and glomerular hypertrophy. The terminal biological half-time of the protein was 3.38±1.87 h after single administration of 500 nmol/kg of HSA-CP in Wister rats. The pharmacokinetic analysis of the protein indicate its promising application in clinical medicine. Key words : C-peptide, human serum albumin, recombinant fusion protein, Pichia pastoris, bioactivity,biological half-time.

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.

Elimination of the free sulfhydryl group in the human serum albumin (HSA) moiety of human interferon-α2b and HSA fusion protein increases its stability against mechanical and thermal stresses

Interferon-α2b (IFN-α2b) and human serum albumin (HSA) fusion protein (IFN-α2b–HSA) is a promising long acting formulation of IFN-α2b for the treatment of hepatitis C. However, accelerated mechanical and thermal stress tests revealed that IFN-α2b–HSA was prone to disulfide-linked aggregation. The formation of aggregates was associated with an increase in immunogenicity in mice. The addition of non-ionic surfactant Tween 80 increased the stability of IFN-α2b–HSA against agitation, but its thermal stability was not improved. Moreover, Tween 80 prompted the aggregation of IFN-α2b–HSA during quiescent storage. To increase the stability of IFN-α2b–HSA, the unpaired cysteine residue in this fusion protein was substituted with serine by site-directed mutagenesis. The resultant fusion protein was designated as IFN-α2b–HSA(C34S). IFN-α2b–HSA(C34S) had significant higher stability over IFN-α2b–HSA, which was evidenced by the facts that after agitation for 72 h or incubation at 60 °C for 2 h, more than 90% of IFN-α2b–HSA(C34S) remained monomeric. Consistent with its improved stability, the immunogenicity of IFN-α2b–HSA(C34S) increased less significantly after agitation. Pharmacokinetics studies in rats revealed that both fusion proteins had similar pharmacokinetic behavior, both with a half-life of about 50 h.

Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagon-like peptide-1 mutants in Pichia pastoris

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. In the present study, overlapping PCR technology was employed to construct two GLP-1 mutants (GLP-1 A2G) 2 and human albumin (HSA) genes in vitro without linker. The spliced gene, (GLP-1 A2G) 2-HSA, was over expressed under the control of promoter AOX1 and Mat α signal peptide in Pichia pastoris. SDS–PAGE and Western blotting were applied to assay the recombinant fusion protein in the culture broth. The results demonstrated that the recombinant (GLP-1 A2G) 2-HSA concentration in the broth could reach a level of 245.0 mg/L and the expressed fusion protein was capable of cross-reacting with anti-human GLP-1 and anti-human albumin antibody. The recombinant (GLP-1 A2G) 2-HSA protein was purified by ultrafiltration, columns of Q-sepharose fast flow and Superdex 75 size-exclusion. The recombinant (GLP-1 A2G) 2-HSA protein obtained could lower in vivo glucose concentration in blood and stimulate in vitro islet cell proliferation. In mouse model, the fusion protein was detectable in plasma even 308 h after a single subcutaneous dose of 1.25 mg/kg. The result showed that the terminal biological half-time of the protein was about 54.2 h which is 650-fold longer than that of GLP-1. The pharmacokinetic analysis of the protein suggests its promising application in clinical medicine.

One-Step Purification of a Fusion Protein of Glucagon-Like Peptide-1 and Human Serum Albumin Expressed inPichia pastorisby an Immunomagnetic Separation Technique

Glucagon-like peptide-1 (GLP-1) has great therapeutic potential to treat diabetes type 2, mainly due to its unique glucose-dependent stimulation of insulin secretion profiles, but its clinical application is limited by its short half-life in vivo, which resultes from degradation by dipeptidyl peptidase IV and/or renal clearance. Developing long-acting GLP-1 analogs is therefore an important step toward using them therapeutically. In this study, the GLP-1/human serum albumin (HSA) fusion protein gene was cloned into the secretor type expression vector pPIC9K and subsequently expressed in Pichia pastoris. The expression quantity reached 58.5 mg/l in small-scale incubation. After optimization and characterization, the GLP-1/HSA fusion protein was successfully purified from the supernatant of the broth using immunomagnetic cellulose microspheres. HPLC showed that the purified GLP-1/HSA had an overall purity of 93.9%, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the fusion protein exhibited the expected molecular mass of 70 kDa. Furthermore, that analysis of in vivo activity indicated that GLP-1/HSA reduced the blood glucose level after intraperitoneal administration to Chinese Kunming mice in a dose-dependent manner, and the effects held significantly 4 h after administration. Overall, this study illustrates the development of a long-acting GLP-1/HSA fusion protein expressed in Pichia pastoris.

Preparation and characterization of a novel exendin‐4 human serum albumin fusion protein expressed in Pichia pastoris

A novel recombinant exendin-4 human serum albumin fusion protein (rEx-4/HSA) expressed in Pichia pastoris was prepared and characterized. Ex-4 is a 39-amino acid peptide isolated from the salivary gland of the lizard Heloderma suspectum and is thought to be a novel therapeutic agent for type 2 diabetes. But to gain a continued effect, the peptide has to be injected twice a day owing to its short plasma half-life (t(1/2) = 2.4 h). To extend the half-life of Ex-4 molecule in vivo, we designed a genetically engineered Ex-4/HSA fusion protein. Between Ex-4 and HSA, a peptide linker GGGGS was inserted and the fusion protein was expressed in methylotrophic yeast P. pastoris with native HSA secretion signal sequence. The recombinant protein was secreted correctly and was obtained with high purity (typically > 98%) by a three-step purification procedure. cAMP assay demonstrated that the fusion protein had a bioactivity similar to Ex-4 for interaction with GLP-1 receptors in vitro. Results from oral glucose tolerance test indicated that rEx-4/HSA could effectively improve glucose tolerance in diabetic db/db mice. Pharmacokinetics studies in cynomologus monkeys also showed that rEx-4/HSA had a much longer plasma half-life. Therefore, rEx-4/HSA fusion protein could potentially be used as a new recombinant biodrug for type 2 diabetes therapy.

Improved Pharmacokinetics of Recombinant Bispecific Antibody Molecules by Fusion to Human Serum Albumin

Recombinant bispecific antibodies such as tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb) have shown to be able to retarget T lymphocytes to tumor cells, leading to their destruction. However, therapeutic efficacy is hampered by a short serum half-life of these small molecules having molecule masses of 50-60 kDa. Thus, improvement of the pharmacokinetic properties of small bispecific antibody formats is required to enhance efficacy in vivo. In this study, we generated several recombinant bispecific antibody-albumin fusion proteins and analyzed these molecules for biological activity and pharmacokinetic properties. Three recombinant antibody formats were produced by fusing two different scFv molecules, bispecific scDb or taFv molecules, respectively, to human serum albumin (HSA). These constructs (scFv(2)-HSA, scDb-HSA, taFv-HSA), directed against the tumor antigen carcinoembryonic antigen (CEA) and the T cell receptor complex molecule CD3, retained full binding capacity to both antigens compared with unfused scFv, scDb, and taFv molecules. Tumor antigen-specific retargeting and activation of T cells as monitored by interleukin-2 release was observed for scDb, scDb-HSA, taFv-HSA, and to a lesser extent for scFv(2)-HSA. T cell activation could be further enhanced by a target cell-specific costimulatory signal provided by a B7-DbCEA fusion protein. Furthermore, we could demonstrate that fusion to serum albumin strongly increases circulation time of recombinant bispecific antibodies. In addition, our comparative study indicates that single chain diabody-albumin fusion proteins seem to be the most promising format for further studying cytotoxic activities in vitro and in vivo.

Characteristics of Saccharomyces cerevisiae gal1Δ and gal1Δhxk2Δ mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.