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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

a Double digestion of the Romiplostim plasmid with BamHI/NdeI, Romiplostim fragment is identified; b Double digestion of pET-22b(+) with BamHI/NdeI
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Fig2: a Double digestion of the Romiplostim plasmid with BamHI/NdeI, Romiplostim fragment is identified; b Double digestion of pET-22b(+) with BamHI/NdeI

Mentions: There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well known genetics and easy genetic manipulation. Production of recombinant protein in E. coli is less costly than using other hosts and the handling is also easier [14]. Therefore the synthetic fragment was successfully transformed into DH5α E. coli competent cells. Colony PCR with M13 primers beside HindIII restriction enzyme assay, showed expected bands at 1000 and 800 bp, respectively (Fig. 1a, b). In this study insoluble form of Romiplostim as inclusion body was assessed and pET22b(+) vector was used. This vector harbor secretion signal and 6xHis fusion sequences, which were omitted from the vector and 6xHis sequences has situated after the stop codon. Amplified fragment and pET-22b(+) plasmid vector were purified and digested with BamHI/NdeI. The fragments were gel purified and subjected to ligation (Fig. 2a, b). After transformation into BL21 (DE3), colony PCR with T7 universal primers was carried out for the retrieved correct colons. The expected size was about 1000 bp (Fig. 3).Fig. 1


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

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

a Double digestion of the Romiplostim plasmid with BamHI/NdeI, Romiplostim fragment is identified; b Double digestion of pET-22b(+) with BamHI/NdeI
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: a Double digestion of the Romiplostim plasmid with BamHI/NdeI, Romiplostim fragment is identified; b Double digestion of pET-22b(+) with BamHI/NdeI
Mentions: There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well known genetics and easy genetic manipulation. Production of recombinant protein in E. coli is less costly than using other hosts and the handling is also easier [14]. Therefore the synthetic fragment was successfully transformed into DH5α E. coli competent cells. Colony PCR with M13 primers beside HindIII restriction enzyme assay, showed expected bands at 1000 and 800 bp, respectively (Fig. 1a, b). In this study insoluble form of Romiplostim as inclusion body was assessed and pET22b(+) vector was used. This vector harbor secretion signal and 6xHis fusion sequences, which were omitted from the vector and 6xHis sequences has situated after the stop codon. Amplified fragment and pET-22b(+) plasmid vector were purified and digested with BamHI/NdeI. The fragments were gel purified and subjected to ligation (Fig. 2a, b). After transformation into BL21 (DE3), colony PCR with T7 universal primers was carried out for the retrieved correct colons. The expected size was about 1000 bp (Fig. 3).Fig. 1

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