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

Immunoblottig pattern of the purified recombinant Romiplostim. Lane 1: Monomeric structure of the recombinant Romiplostim (30 kDa), disulfide bonds are reduced with 2ME; Lane 2: Dimeric structure of recombinant Romiplostim (60 kDa), 2ME was omitted of the loading buffer. The findings showed accurate molecules were produced and purified
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Immunoblottig pattern of the purified recombinant Romiplostim. Lane 1: Monomeric structure of the recombinant Romiplostim (30 kDa), disulfide bonds are reduced with 2ME; Lane 2: Dimeric structure of recombinant Romiplostim (60 kDa), 2ME was omitted of the loading buffer. The findings showed accurate molecules were produced and purified

Mentions: In contrast to US patent (6835809 B1) which researchers recruited two step Ion Exchange chromatography, in the present study we used protein A affinity chromatography system to reduce purification stages. In order to perceive refolded and purified recombinant Romiplostim by protein A affinity chromatography, immunoblotting was performed (Fig. 6); 2ME was omitted of the loading buffer, so 60 kDa protein band was shown which was related to the quaternary structure of this protein. Besides, 30 kDa protein band in the form of denaturated protein was observed (loading buffer contained 2ME). These findings showed accurate molecules were produced and purified.Fig. 6


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

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

Immunoblottig pattern of the purified recombinant Romiplostim. Lane 1: Monomeric structure of the recombinant Romiplostim (30 kDa), disulfide bonds are reduced with 2ME; Lane 2: Dimeric structure of recombinant Romiplostim (60 kDa), 2ME was omitted of the loading buffer. The findings showed accurate molecules were produced and purified
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Immunoblottig pattern of the purified recombinant Romiplostim. Lane 1: Monomeric structure of the recombinant Romiplostim (30 kDa), disulfide bonds are reduced with 2ME; Lane 2: Dimeric structure of recombinant Romiplostim (60 kDa), 2ME was omitted of the loading buffer. The findings showed accurate molecules were produced and purified
Mentions: In contrast to US patent (6835809 B1) which researchers recruited two step Ion Exchange chromatography, in the present study we used protein A affinity chromatography system to reduce purification stages. In order to perceive refolded and purified recombinant Romiplostim by protein A affinity chromatography, immunoblotting was performed (Fig. 6); 2ME was omitted of the loading buffer, so 60 kDa protein band was shown which was related to the quaternary structure of this protein. Besides, 30 kDa protein band in the form of denaturated protein was observed (loading buffer contained 2ME). These findings showed accurate molecules were produced and purified.Fig. 6

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