Cloning, Optimization of Periplasmic Expression and Purification of Recombinant Granulocyte Macrophage-Stimulating Factor in BL21 (DE3)

Overview

Journal Adv Biomed Res

Specialty Biomedical Engineering

Date 2020 Feb 1

PMID 32002394

Citations 3

Authors

Elham Taherian

Elmira Mohammadi

Ali Jahanian-Najafabadi

Fatemeh Moazen

Vajihe Akbari

Affiliations

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Abstract

Background: Molgramostim, a nonglycosylated version of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), can be produced in a high level by . However, overexpression of GM-CSF in bacterial cells usually leads to formation of inclusion bodies and insoluble protein aggregates which are not biologically active. The aim of the present study was to improve the expression of soluble and biologically active GM-CSF in periplasmic space of BL21 (DE3).

Materials And Methods: The codon-optimized GM-CSF gene was subcloned into pET-22b expression vector, in frame with the secretion signal peptide for periplasmic secretion. Cultivation conditions including as isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, incubation temperature, and presence of sucrose were optimized to improve periplasmic expression of GM-CSF. The expressed protein was purified using Ni-NTA affinity column. Biological activity of GM-CSF on HL-60 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Results: The amount of soluble protein for periplasmic expression was more when compared with one of the cytoplasmic expressions. The optimum condition for periplasmic expression of GM-CSF was expression at 23°C, using 1 mM IPTG as inducer and in the presence of 0.4 M sucrose. The biological activity of purified GM-CSF on HL-60 cell line was assessed by MTT assay, and the specific activity of produced GM-CSF was determined as 1.2 × 10 IU/μg.

Conclusion: The present work suggests that periplasmic expression and optimization of cultivation conditions could improve soluble expression of recombinant proteins by .

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