STUDIES ON SULFATION FACTOR (SF) ACTIVITY OF HUMAN SERUM

Human serum albumin (HSA) is the most abundant protein in blood and has a 19-day in vivo half-life, the longest human blood protein. HSA has also been extensively studied as a drug carrier in a wide variety of clinical applications. HSA-binding, compared with HSA-fusion, is promising strategy for extending the plasma half-life of protein therapeutics. The construction of albumin-binding drugs requires assessment of a large enough quantity of HSA-binding peptide candidates for conjugation with therapeutic proteins. Here, we report a back-of-the-envelope assessment method to facilitate phage display selection of HSA-binding peptides. With an experimentally determined number of phage titers, we can calculate the specificity ratios and the recovery yields. The recovery yield is calculated using the titers of eluted phage divided by the titers of input phage. The specificity ratio is calculated using the titer of eluted phage from a target-coated plate divided by the titer of eluted phage from a blank-control plate. These parameters are defined as quantitative criteria for panning and characterization of binding phage clones. Consequently, this approach may enable more rapid and low-cost phage display screening of HSA-binding peptides, which could be used as candidates of HSA binders for conjugation with therapeutic proteins.

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Temperature dependence of certain integrated membrane functions in macrophages

Journal of Cell Science ◽

10.1242/jcs.57.1.115 ◽

1982 ◽

Vol 57 (1) ◽

pp. 115-127

Author(s):

M Faghihi Shirazi ◽

N.N. Aronson ◽

R.T. Dean

Keyword(s):

Human Serum Albumin ◽

Bovine Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Half Life ◽

Bovine Serum ◽

Fluid Phase ◽

Peritoneal Macrophages ◽

Turnover Time ◽

Effect Of Temperature

We have studied the effect of temperature on uptake and degradation of molecules entering mouse peritoneal macrophages by fluid-phase, adsorptive and receptor-mediated pinocytosis, and on degradation of their intracellular proteins. Uptake of [3H]sucrose and uptake and degradation of formaldehyde-treated 125I-labelled human serum albumin and 125I-labelled mannose-bovine serum albumin continued, but were progressively slowed as the temperature decreased from 37 degrees C to 20 degrees C. The uptake and degradation were completely abolished at approximately 15 degrees C. Arrhenius plots for adsorptive and fluid uptake were unilinear, whereas that for receptor-mediated endocytosis showed an inflection point at approximately 20 degrees C. The results did not indicate any distinction between adsorptive and fluid pinocytosis. An ‘intracellular turnover time’ calculated for mannose-bovine serum albumin taken up by the specific route is 19–24 min and this time calculated for human serum albumin is, in contrast, 99 min. Studies of the kinetics of degradation of both endocytosed and endogenous proteins showed similarity in the temperature cut-off of degradation of endocytosed and endogenous long-half-life proteins (congruent to 15 degrees C) and continuance of endogenous short-half-life degradation at much lower temperatures.

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A novel exendin-4 human serum albumin fusion protein, E2HSA, with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2014.02.045 ◽

2014 ◽

Vol 445 (2) ◽

pp. 511-516 ◽

Cited By ~ 12

Author(s):

Ling Zhang ◽

Lin Wang ◽

Zhiyun Meng ◽

Hui Gan ◽

Ruolan Gu ◽

...

Keyword(s):

Human Serum Albumin ◽

Fusion Protein ◽

Serum Albumin ◽

Human Serum ◽

Half Life ◽

Rhesus Monkeys

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Human Growth Hormone-Releasing Factor (hGRF)1–29-Albumin Bioconjugates Activate the GRF Receptor on the Anterior Pituitary in Rats: Identification of CJC-1295 as a Long-Lasting GRF Analog

Endocrinology ◽

10.1210/en.2004-1286 ◽

2005 ◽

Vol 146 (7) ◽

pp. 3052-3058 ◽

Cited By ~ 27

Author(s):

Lucie Jetté ◽

Roger Léger ◽

Karen Thibaudeau ◽

Corinne Benquet ◽

Martin Robitaille ◽

...

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Half Life ◽

Anterior Pituitary ◽

Ex Vivo ◽

Normal Male ◽

Pituitary Cells ◽

Sprague Dawley ◽

Plasma Half Life

Abstract In vivo bioconjugation to the free thiol on Cys34 of serum albumin by a strategically placed reactive group on a bioactive peptide is a useful tool to extend plasma half-life. Three maleimido derivates of human GH-releasing factor (hGRF)1–29 were synthesized and bioconjugated to human serum albumin ex vivo. All three human serum albumin conjugates showed enhanced in vitro stability against dipeptidylpeptidase-IV and were bioactive in a GH secretion assay in cultured rat anterior pituitary cells. When the maleimido derivatives were individually administered sc to normal male Sprague Dawley rats, an acute secretion of GH was measured in plasma. The best compound, CJC-1295, showed a 4-fold increase in GH area under the curve over a 2-h period compared with hGRF1–29. CJC-1295, a tetrasubstituted form of hGRF1–29 with an added Nε-3-maleimidopropionamide derivative of lysine at the C terminus, was selected for further pharmacokinetic evaluation, where it was found to be present in plasma beyond 72 h. A Western blot analysis of the plasma of a rat injected with CJC-1295 showed the presence of a CJC-1295 immunoreactive species on the band corresponding to serum albumin, appearing after 15 min and remaining in circulation beyond 24 h. These results led to the identification of CJC-1295 as a stable and active hGRF1–29 analog with an extended plasma half-life.

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Extending the Serum Half-Life of G-CSF via Fusion with the Domain III of Human Serum Albumin

BioMed Research International ◽

10.1155/2013/107238 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

Shuqiang Zhao ◽

Yu Zhang ◽

Hong Tian ◽

Xiaofei Chen ◽

Di Cai ◽

...

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Half Life ◽

Fusion Gene ◽

Recombinant Expression ◽

Pharmacokinetic Studies ◽

Protein Fusion ◽

Reading Frame ◽

Domain Iii

Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF), the domain III of human serum albumin (3DHSA) was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned intopPICZαA along with the open reading frame of theα-factor signal under the control of the AOX1 promoter. The recombinant expression vector was transformed intoPichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC) counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF.

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Rapid identification of Human Serum Albumin binding peptides from a phage display library：a back-of-the-envelope assessment of positive binders using titer-based criteria

10.7287/peerj.preprints.1115v1 ◽

2015 ◽

Author(s):

Yi-Feng Shi ◽

Min Li ◽

Jia-Di Zhang ◽

Lei Bian

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Phage Display ◽

Human Serum ◽

Half Life ◽

Drug Carrier ◽

Therapeutic Proteins ◽

Blood Protein ◽

Albumin Binding ◽

Binding Peptides

Human serum albumin (HSA) is the most abundant protein in blood and has a 19-day in vivo half-life, the longest human blood protein. HSA has also been extensively studied as a drug carrier in a wide variety of clinical applications. HSA-binding, compared with HSA-fusion, is promising strategy for extending the plasma half-life of protein therapeutics. The construction of albumin-binding drugs requires assessment of a large enough quantity of HSA-binding peptide candidates for conjugation with therapeutic proteins. Here, we report a back-of-the-envelope assessment method to facilitate phage display selection of HSA-binding peptides. With an experimentally determined number of phage titers, we can calculate the specificity ratios and the recovery yields. The recovery yield is calculated using the titers of eluted phage divided by the titers of input phage. The specificity ratio is calculated using the titer of eluted phage from a target-coated plate divided by the titer of eluted phage from a blank-control plate. These parameters are defined as quantitative criteria for panning and characterization of binding phage clones. Consequently, this approach may enable more rapid and low-cost phage display screening of HSA-binding peptides, which could be used as candidates of HSA binders for conjugation with therapeutic proteins.

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