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

Sepideh  Abbaszadeh; Shahin  Eghbalsaied; Meysam  Soleimani; Sadegh  Khazalpour; Saeid  Afshar

doi:10.18502/ajmb.v17i2.18563

Sepideh Abbaszadeh Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Hamadan University of Medical Sciences, Hamadan, Iran
Shahin Eghbalsaied Research Center for Molecular Medicine, Institute of Cancer, Hamadan University of Medical Sciences, Hamadan, Iran
Meysam Soleimani Department of Pharmaceutical Biotechnology, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran
Sadegh Khazalpour Department of Analytical Chemistry, Faculty of Chemistry and Petroleum Science, Bu-Ali Sina University, Hamadan, Iran
Saeid Afshar Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Hamadan University of Medical Sciences, Hamadan, Iran

DOI: https://doi.org/10.18502/ajmb.v17i2.18563

Keywords: Base sequence, CRISPR-associated proteins, Escherichia coli, Protein biosynthesis

Abstract

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-thiogalacto-pyranoside (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.