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Volume 32, Issue 155 (November & December 2024)
J Adv Med Biomed Res 2024, 32(155): 438-448 |
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Mobasheri T, Sharafi A, Mohit E. Evaluating the Effects of Environmental and Culture Conditions on Anti-HER2 scFv Expression and Solubility in E. coli. J Adv Med Biomed Res 2024; 32 (155) :438-448
URL: http://journal.zums.ac.ir/article-1-7604-en.html
URL: http://journal.zums.ac.ir/article-1-7604-en.html
1- , el_mohit@yahoo.com
Abstract: (79 Views)
Background & Objective: Breast cancer is an international concern due to its high prevalence worldwide. Human epidermal growth factor receptor 2 (HER2)-positive breast cancers were observed in 15-20% of breast cancers, with higher aggressiveness and poor prognosis. The single-chain fragment variable (scFv) structure includes variable regions of antibody light and heavy chains. Despite the promising potential of anti-HER2 scFv for non-invasive detection of HER2 status, its low expression and solubility remain significant challenges. Here, we intended to optimize the anti-HER2 scFv expression and solubility in BL21-pET-22 (anti-HER2 scFv).
Materials & Methods: The effects of ethanol stress, aeration, post-induction temperatures, induction-starting times, culture mediums, and MgCl2 addition were examined.
Results: We observed that ethanol stress (1% v/v) increased protein expression and decreased protein solubility at 37 °C. However, it enhanced anti-HER2 scFv solubility at lower post-induction temperature (22 °C). Induction at OD600=1 elevated anti-HER2 scFv expression, along with a substantial protein solubility enhancement at OD600=0.6-0.8. In the LB medium, speeding up the shakers to 250 RPM led to a non-significant enhancement of anti-HER2 expression and a significant solubility improvement. The best medium for the optimum anti-HER2 scFv expression and solubility was the TB medium. MgCl2 could not increase anti-HER2 expression or solubility.
Conclusion: The highest anti-HER2 scFv expression level was obtained when BL21-pET-22 (anti-HER2 scFv) was cultured in TB medium, induced at OD600=2 and 37 °C, and incubated at 250 RPM. The highest solubility was also observed in the TB medium, induced at OD600=0.6-0.8 and 37 °C, and incubated at 250 RPM.
Materials & Methods: The effects of ethanol stress, aeration, post-induction temperatures, induction-starting times, culture mediums, and MgCl2 addition were examined.
Results: We observed that ethanol stress (1% v/v) increased protein expression and decreased protein solubility at 37 °C. However, it enhanced anti-HER2 scFv solubility at lower post-induction temperature (22 °C). Induction at OD600=1 elevated anti-HER2 scFv expression, along with a substantial protein solubility enhancement at OD600=0.6-0.8. In the LB medium, speeding up the shakers to 250 RPM led to a non-significant enhancement of anti-HER2 expression and a significant solubility improvement. The best medium for the optimum anti-HER2 scFv expression and solubility was the TB medium. MgCl2 could not increase anti-HER2 expression or solubility.
Conclusion: The highest anti-HER2 scFv expression level was obtained when BL21-pET-22 (anti-HER2 scFv) was cultured in TB medium, induced at OD600=2 and 37 °C, and incubated at 250 RPM. The highest solubility was also observed in the TB medium, induced at OD600=0.6-0.8 and 37 °C, and incubated at 250 RPM.
Keywords: Breast Neoplasms, Antibody Fragments, HER2 (Receptor, ErbB-2), Protein expression, Solubility
Type of Study: Original Article |
Subject:
Bionanotechnology
Received: 2024/11/12 | Accepted: 2025/02/1 | Published: 2024/10/10
Received: 2024/11/12 | Accepted: 2025/02/1 | Published: 2024/10/10
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