Evaluating the Effects of Environmental and Culture Conditions on Anti-HER۲ scFv Expression and Solubility in E. coli

Background & Objective:  Breast cancer is an international concern due to its high prevalence worldwide. Human epidermal growth factor receptor ۲ (HER۲)-positive breast cancers were observed in ۱۵-۲۰% 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-HER۲ scFv for non-invasive detection of HER۲ status, its low expression and solubility remain significant challenges. Here, we intended to optimize the anti-HER۲ scFv expression and solubility in BL۲۱-pET-۲۲ (anti-HER۲ scFv).  Materials & Methods:  The effects of ethanol stress, aeration, post-induction temperatures, induction-starting times, culture mediums, and MgCl۲ addition were examined. Results:  We observed that ethanol stress (۱% v/v) increased protein expression and decreased protein solubility at ۳۷ °C. However, it enhanced anti-HER۲ scFv solubility at lower post-induction temperature (۲۲ °C). Induction at OD۶۰۰=۱ elevated anti-HER۲ scFv expression, along with a substantial protein solubility enhancement at OD۶۰۰=۰.۶-۰.۸. In the LB medium, speeding up the shakers to ۲۵۰ RPM led to a non-significant enhancement of anti-HER۲ expression and a significant solubility improvement. The best medium for the optimum anti-HER۲ scFv expression and solubility was the TB medium. MgCl۲ could not increase anti-HER۲ expression or solubility. Conclusion:  The highest anti-HER۲ scFv expression level was obtained when BL۲۱-pET-۲۲ (anti-HER۲ scFv) was cultured in TB medium, induced at OD۶۰۰=۲ and ۳۷ °C, and incubated at ۲۵۰ RPM. The highest solubility was also observed in the TB medium, induced at OD۶۰۰=۰.۶-۰.۸ and ۳۷ °C, and incubated at ۲۵۰ RPM.

کلیدواژه ها:

Breast Neoplasms ، Antibody Fragments ، HER۲ (Receptor ، ErbB-۲) ، Protein expression ، Solubility