Recombinant production of mecasermin in E. coli expression system

Area of Science:

• Biotechnology and Recombinant Protein Production
• Molecular Biology and Genetic Engineering
• Protein Chemistry and Therapeutics

Background:

• Human Insulin-like Growth Factor 1 (hIGF-1) is a 70-amino acid protein with therapeutic applications, but engineered variants like mecasermin offer improved properties.
• Mecasermin (T4I substitution) is an approved therapeutic for conditions including growth failure and amyotrophic lateral sclerosis, yet its recombinant production in E. coli was uninvestigated.
• Efficient recombinant production systems are crucial for scaling up therapeutic protein manufacturing.

Purpose of the Study:

• To investigate and establish an efficient Escherichia coli (E. coli) expression system for the recombinant production of mecasermin.
• To compare the expression levels of mecasermin in two different E. coli strains, Origami B (DE3) and BL21 (DE3).
• To develop a purification strategy for recombinant mecasermin produced as inclusion bodies.

Main Methods:

• Designed and optimized the mecasermin DNA sequence for E. coli codon preference and cloned it into the pET15b vector.
• Transformed E. coli Origami B (DE3) and BL21 (DE3) with the recombinant vector and induced protein expression.
• Confirmed protein identity and purity using Western blotting and mass spectrometry, followed by purification via gel filtration chromatography.

Main Results:

• E. coli Origami B (DE3) exhibited approximately twice the expression level of mecasermin compared to E. coli BL21 (DE3).
• Recombinant mecasermin was successfully overexpressed and confirmed by molecular analysis techniques.
• A purification protocol involving isolation and refolding of inclusion bodies, followed by gel filtration, yielded purified recombinant mecasermin.

Conclusions:

• E. coli Origami B (DE3) is a suitable and preferable host for high-yield recombinant mecasermin production.
• This study provides the first report on the recombinant production of mecasermin in an E. coli expression system.
• The developed purification method enables the successful isolation of functional recombinant mecasermin for potential therapeutic use.