Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

The recombinant human granulocyte colony stimulating factor conjugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony...

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Bibliographic Details
Published inAvicenna journal of medical biotechnology Vol. 13; no. 4; pp. 192 - 200
Main Authors Trinh, Nguyen Thi My, Thuoc, Tran Linh, Thao, Dang Thi Phuong
Format Journal Article
LanguageEnglish
Published Iran Avicenna Research Institute 01.10.2021
Subjects
Analysis
Cancer
Chemotherapy
Chromatography
Escherichia coli
Ethylenediaminetetraacetic acid
Fermentation
Original
Polyethylene glycol
Polyols
Vietnam
Polyethylene glycol
Granulocyte colony stimulating factor (G-CSF)
Inclusion bodies
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Summary:The recombinant human granulocyte colony stimulating factor conjugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimulating Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 bioreactor and the expression of GCSF was induced by adding 0.5 IPTG. After 24 of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the conjugation with 5 PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. PEGylated GCSF was obtained with high purity (∼97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 PEG molecule (monoPEG-GCSF). These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in
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ISSN:2008-2835
2008-4625
DOI:10.18502/ajmb.v13i4.7204