Avicenna J Med Biotech

arij002

Avicenna Journal of Medical Biotechnology

2008-2835 2008-4625

Avicenna Research Institute

ajmb60610

Optimization of RfxCas13d Expression in Escherichia coli Host using Response Surface Methodology

Abbaszadeh

Sepideh

Eghbalsaied

Shahin

Soleimani

Meysam

Khazalpour

Sadegh

Afshar

Saeid

17 2 122 130 4 9 2024 4 3 2025

Background: RfxCas13d, a key member of the Cas13 family, plays a vital role in CRISPR-based diagnostics for RNA sequence detection and gene silencing. This study aimed to enhance RfxCas13d expression by optimizing key parameters using Response Surface Methodology (RSM). Methods: The plasmid pET28b-RfxCas13d-His (Addgene 141322) was introduced into BL21 (DE3) and Rosetta™ (DE3) strains. Initial expression tests were conducted, followed by RSM-guided optimization of factors such as isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, temperature, cell density at induction, and induction time in BL21 (DE3). Protein expression levels were quantified using ImageJ and AlphaEaseFC software to analyze band intensities. Results: BL21 (DE3) was selected for further optimization based on preliminary results. Analysis of 26 RSM-designed experiments revealed that temperature, induction time, IPTG concentration, and their interactions significantly influenced RfxCas13d expression. Optimal conditions were identified as 0.25 mM IPTG, an OD600 nm of 0.8 at induction, 37°C, and Overnight (ON) of induction. The regression model exhibited high accuracy, with a correlation coefficient of 0.97 and a p-value less than 0.05, confirming a strong linear relationship between predicted and observed values. Conclusion: This study highlights the significant impact of the four optimized factors on RfxCas13d expression. Under optimized conditions, a soluble protein concentration of 3.6 mg/100 ml cell culture was achieved after purification. It represents the first application of RSM for optimizing RfxCas13d expression, providing a foundation for further refinement of expression conditions. Continued use of RSM in future research will enhance the efficiency of RfxCas13d production for diagnostic and therapeutic applications.