Improving the Expression of Human Granulocyte Colony Stimulating Factor in Escherichia coli by Reducing the GC-content and Increasing mRNA Folding Free Energy at 5’-Terminal End

Dewi, Kartika Sari and Fuad, Asrul Muhamad (2020) Improving the Expression of Human Granulocyte Colony Stimulating Factor in Escherichia coli by Reducing the GC-content and Increasing mRNA Folding Free Energy at 5’-Terminal End. Advanced Pharmaceutical Bulletin, 10 (4). pp. 610-616. ISSN 2228-5881

[thumbnail of apb-10-610.pdf] Text
apb-10-610.pdf - Published Version

Download (601kB)

Abstract

Purpose: Strategy for improving the production of biopharmaceutical protein continues to develop due to increasing market demand. Human granulocyte colony stimulating factor (hG-CSF) is one of biopharmaceutical proteins that has many applications, and easily produced in Escherichia coli expression system. Previous studies reported that codon usage, rare codon, mRNA folding and GC-content at 5’-terminal end were crucial for protein production in E. coli. In the present study, the effect of reducing the GC-content and increasing the mRNA folding free energy at the 5’-terminal end on the expression level of hG-CSF proteins was investigated.

Methods: Synonymous codon substitutions were performed to generate mutant variants of open reading frame (ORF) with lower GC-content at 5’-terminal ends. Oligoanalyzer tool was used to calculate the GC content of eight codons sequence after ATG. Whereas, mRNA folding free energy was predicted using KineFold and RNAfold tools. The template DNA was amplified using three variant forward primers and one same reverse primer. Those DNA fragments were individually cloned into pJexpress414 expression vector and were confirmed using restriction and DNA sequencing analyses. The confirmed constructs were transformed into E. coli NiCo21(DE3) host cells and the recombinant protein was expressed using IPTG-induction. Total protein obtained were characterized using SDS-PAGE, Western blot and ImageJ software analyses.

Results: The result showed that the mutant variant with lower GC-content and higher mRNA folding free energy near the translation initiation region (TIR) could produce a higher amount of hG-CSF proteins compared to the original gene sequence.

Conclusion: This study emphasized the important role of the nucleotide composition immediately downstream the start codon to achieve high-yield protein product on heterologous expression in E. coli.

Item Type: Article
Subjects: Universal Eprints > Medical Science
Depositing User: Managing Editor
Date Deposited: 12 Apr 2023 04:37
Last Modified: 30 Jan 2024 06:19
URI: http://journal.article2publish.com/id/eprint/1671

Actions (login required)

View Item
View Item