Limits...
Expression, purification and biological activity assessment of romiplostim biosimilar peptibody.

Fayaz S, Fard-Esfahani P, Golkar M, Allahyari M, Sadeghi S - Daru (2016)

Bottom Line: Designed construct in E. coli host resulted in protein expression in cytoplasm as inclusion body.The resulting peptibodies were characterized by SDS-PAGE and Western immunoblotting.This new approach in expression and purification of this recently introduced thrombopoietin receptor agonist drug may be followed by scale up of its production to response the chronic ITP patient's demand.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

ABSTRACT

Background: Romiplostim is a peptibody analogue of thrombopoietin (TPO) which regulates platelet production. This molecule consists of two main parts: Peptide sequences which like wild type TPO, mimics stimulation of TPO receptor and IgG1Fc, (Peptide + Antibody = Peptibody). This drug is used in treatment of chronic Immune Thrombocytopenic Purpura (ITP).

Methods: In this project E. coli bacteria were transformed by a construct harboring peptibody fusion gene. This construct consisted of two repeated peptide sequences which have fused to Carboxyl group of IgG1Fc. Designed construct in E. coli host resulted in protein expression in cytoplasm as inclusion body. The inclusion bodies were separated, washed and after denaturation and solubilization, in the last stage the desired peptibodies were refolded and purified. The resulting peptibodies were characterized by SDS-PAGE and Western immunoblotting. The bioactivity were assessed in vivo using subcutaneous injection in mice.

Results: Results showed accurate molecules were produced and purified. Also, in vivo experiment showed significant increment (more than two fold) of platelets compared to control group.

Conclusion: In this study laboratory scale production of recombinant romiplostim showed proper in-vivo bioactivity. This new approach in expression and purification of this recently introduced thrombopoietin receptor agonist drug may be followed by scale up of its production to response the chronic ITP patient's demand.

No MeSH data available.


Related in: MedlinePlus

SDS-PAGE analysis of 2 h, 4 h, 6 h and overnight induced with 1 mM IPTG beside overnight uninduced of recombinant Romiplostim. High expression of Romiplostim inclusion body in uninduced recombinant Romiplostim was observed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4940717&req=5

Fig4: SDS-PAGE analysis of 2 h, 4 h, 6 h and overnight induced with 1 mM IPTG beside overnight uninduced of recombinant Romiplostim. High expression of Romiplostim inclusion body in uninduced recombinant Romiplostim was observed

Mentions: Expressed recombinant protein of induced and uninduced cells, was compared (Fig. 4), high expression of Romiplostim inclusion body in the absence of IPTG was a great cost effective point. In Western blot, Fcγ carrier domain of the recombinant protein was reactive to goat anti-human IgG Fcγ as a primary antibody, followed by peroxidase conjugated rabbit anti-sheep IgG. Sharp and distinct Romiplostim recombinant protein band in 30 kDa was in accordance with the calculated molecular weight of Romiplostim single strand (Fig. 5).Fig. 4


Expression, purification and biological activity assessment of romiplostim biosimilar peptibody.

Fayaz S, Fard-Esfahani P, Golkar M, Allahyari M, Sadeghi S - Daru (2016)

SDS-PAGE analysis of 2 h, 4 h, 6 h and overnight induced with 1 mM IPTG beside overnight uninduced of recombinant Romiplostim. High expression of Romiplostim inclusion body in uninduced recombinant Romiplostim was observed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940717&req=5

Fig4: SDS-PAGE analysis of 2 h, 4 h, 6 h and overnight induced with 1 mM IPTG beside overnight uninduced of recombinant Romiplostim. High expression of Romiplostim inclusion body in uninduced recombinant Romiplostim was observed
Mentions: Expressed recombinant protein of induced and uninduced cells, was compared (Fig. 4), high expression of Romiplostim inclusion body in the absence of IPTG was a great cost effective point. In Western blot, Fcγ carrier domain of the recombinant protein was reactive to goat anti-human IgG Fcγ as a primary antibody, followed by peroxidase conjugated rabbit anti-sheep IgG. Sharp and distinct Romiplostim recombinant protein band in 30 kDa was in accordance with the calculated molecular weight of Romiplostim single strand (Fig. 5).Fig. 4

Bottom Line: Designed construct in E. coli host resulted in protein expression in cytoplasm as inclusion body.The resulting peptibodies were characterized by SDS-PAGE and Western immunoblotting.This new approach in expression and purification of this recently introduced thrombopoietin receptor agonist drug may be followed by scale up of its production to response the chronic ITP patient's demand.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

ABSTRACT

Background: Romiplostim is a peptibody analogue of thrombopoietin (TPO) which regulates platelet production. This molecule consists of two main parts: Peptide sequences which like wild type TPO, mimics stimulation of TPO receptor and IgG1Fc, (Peptide + Antibody = Peptibody). This drug is used in treatment of chronic Immune Thrombocytopenic Purpura (ITP).

Methods: In this project E. coli bacteria were transformed by a construct harboring peptibody fusion gene. This construct consisted of two repeated peptide sequences which have fused to Carboxyl group of IgG1Fc. Designed construct in E. coli host resulted in protein expression in cytoplasm as inclusion body. The inclusion bodies were separated, washed and after denaturation and solubilization, in the last stage the desired peptibodies were refolded and purified. The resulting peptibodies were characterized by SDS-PAGE and Western immunoblotting. The bioactivity were assessed in vivo using subcutaneous injection in mice.

Results: Results showed accurate molecules were produced and purified. Also, in vivo experiment showed significant increment (more than two fold) of platelets compared to control group.

Conclusion: In this study laboratory scale production of recombinant romiplostim showed proper in-vivo bioactivity. This new approach in expression and purification of this recently introduced thrombopoietin receptor agonist drug may be followed by scale up of its production to response the chronic ITP patient's demand.

No MeSH data available.


Related in: MedlinePlus