Volume 33, Issue 2 (5-2025)
JSSU 2025, 33(2): 8705-8717 |
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Hajizadeh M, Jafari Kakhki A, Nabavinia M, Nabavinia M. Cloning and Optimization of Formate Dehydrogenase Gene Expression in E. COLI. JSSU 2025; 33 (2) :8705-8717
URL: http://jssu.ssu.ac.ir/article-1-6174-en.html
URL: http://jssu.ssu.ac.ir/article-1-6174-en.html
Abstract: (14 Views)
Introduction: The increase in greenhouse gas levels and the gradual warming of the earth have garnered worldwide interest. The enzyme formate-dehydrogenase can reduce CO2 by converting it into formate, which is subsequently converted into methanol by the enzyme alcohol dehydrogenase. The purpose of this research was to evaluate the effect of different concentrations of IPGT, modifier, induction time and culture medium on the expression of formate-dehydrogenase enzyme and optimization.
Methods: Specific primers were designed to amplify the gene fragment using polymerase chain reaction (PCR). The Formate-dehydrogenase enzyme gene was inserted into the pET28b vector, and the recombinant plasmid was used to transform E.coli. To enhance protein expression, a Taguchi experimental design was conducted to investigate the effects of temperature, inducer concentration, and type of culture medium type on protein expression levels at two different levels.
Results: PCR results confirmed the amplification of formate-dehydrogenase enzyme gene in E. coli DH5α. Induction time and concentration negatively impacted on expression. The highest level of expression of the recombinant protein was observed 24 hours post-induction at a temperature of 25°C, with an IPTG concentration of 0.1 mM in the presence of sorbitol. LB culture medium was chosen as the best expression medium.
Conclusion: This study showed that low concentrations of IPTG were sufficient for producing the recombinant protein, proving to be economical. Reducing the induction time also increased protein expression. LB medium enhanced with sorbitol was identified to be the best culture medium for inducing the expression of the formate dehydrogenase enzyme.
Methods: Specific primers were designed to amplify the gene fragment using polymerase chain reaction (PCR). The Formate-dehydrogenase enzyme gene was inserted into the pET28b vector, and the recombinant plasmid was used to transform E.coli. To enhance protein expression, a Taguchi experimental design was conducted to investigate the effects of temperature, inducer concentration, and type of culture medium type on protein expression levels at two different levels.
Results: PCR results confirmed the amplification of formate-dehydrogenase enzyme gene in E. coli DH5α. Induction time and concentration negatively impacted on expression. The highest level of expression of the recombinant protein was observed 24 hours post-induction at a temperature of 25°C, with an IPTG concentration of 0.1 mM in the presence of sorbitol. LB culture medium was chosen as the best expression medium.
Conclusion: This study showed that low concentrations of IPTG were sufficient for producing the recombinant protein, proving to be economical. Reducing the induction time also increased protein expression. LB medium enhanced with sorbitol was identified to be the best culture medium for inducing the expression of the formate dehydrogenase enzyme.
Type of Study: Original article |
Subject:
Microbiology
Received: 2024/02/16 | Accepted: 2024/05/29 | Published: 2025/05/5
Received: 2024/02/16 | Accepted: 2024/05/29 | Published: 2025/05/5
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