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Indirect optimization of staphylokinase expression level in dicistronic auto-inducible system
Fatemeh Sadat Shariati1, Malihe Keramati2, Reza Ahangari Cohan3
1Department of Nanobiotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran.
AMB Express
|September 22, 2022
Summary
We optimized staphylokinase (SAK) production using Design of Experiments (DOE). Optimal conditions increased SAK expression fourfold, enabling purification and activity confirmation.
Area of Science:
- Biotechnology
- Molecular Biology
- Biochemistry
Background:
- Recombinant protein production requires optimized expression systems.
- Previous work established a novel Hsp27 SILEX system for protein production.
- Staphylokinase (SAK) is a valuable therapeutic protein.
Purpose of the Study:
- To optimize autoinduction conditions for staphylokinase (SAK) expression.
- To identify key factors influencing SAK production using statistical methods.
- To enhance the yield of recombinant SAK using the Hsp27 SILEX system.
Main Methods:
- Design of Experiments (DOE) with Response Surface Methodology (RSM).
- Optimization of inoculation load, self-induction temperature, and culture media.
- Fluorometry for assaying SAK expression levels.
- Immobilized metal affinity chromatography for protein purification.
Main Results:
- All tested parameters (inoculation load, temperature, media) significantly affected SAK expression (p < 0.05).
- Optimal conditions identified: 0.05 inoculation load, 25°C, and TB culture medium.
- A quadratic model (R² = 0.91) accurately predicted optimal expression.
- Optimized conditions resulted in a fourfold increase in SAK expression (1.3 to 5.2 µg/ml).
Conclusions:
- The Hsp27 SILEX system, optimized via DOE, significantly enhances staphylokinase production.
- The identified optimal conditions provide a robust method for high-yield recombinant SAK expression.
- Purified SAK retained its biological activity, confirmed by caseinolytic assay.
