Expression efficiency of the human thrombomodulin-encoding gene in various vector and host systems

Area of Science:

• Biotechnology
• Molecular Biology
• Genetic Engineering

Background:

• Developing efficient expression systems is crucial for producing recombinant proteins like human thrombomodulin (TM).
• Mammalian expression vectors offer advantages for complex protein production but require careful optimization for different host cells.

Purpose of the Study:

• To evaluate and compare the performance of five mammalian expression vectors for recombinant human TM production.
• To identify optimal expression strategies and promoters for high-yield TM generation in various cell lines.

Main Methods:

• Construction of expression vectors with monocistronic gene cassettes including selectable markers (HyR) and amplifiable genes (Dhfr).
• Transfection and evaluation of vector performance in HEK293, BHK, CHL-1, and CHO/Dhfr- cell lines.
• Analysis of DNA and mRNA levels, transient and stable expression efficiencies, and gene amplification effects.

Main Results:

• Expression vector performance was cell-line dependent regarding DNA uptake, transient, and stable expression.
• The myeloproliferative sarcoma virus (MPSV) LTR promoter consistently yielded higher TM expression levels, especially in HEK293 cells.
• Gene amplification of Dhfr using methotrexate resulted in a 20-fold increase in TM production in CHO/Dhfr- cells.

Conclusions:

• The pMPSV-TM vector exhibits high transcription efficiency and is a promising system for recombinant human TM production.
• Optimization of expression vectors and promoter selection is critical for maximizing protein yield in specific host cell lines.