Prokaryotic Expression, Refolding and Purification of High-Purity Mouse Midkine in Escherichia coli

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Human Midkine shows potential as an articular protective agent.
• Preclinical studies are necessary to evaluate its safety and efficacy.
• A scalable and cost-effective production method for recombinant mouse Midkine (rmMK) is required.

Purpose of the Study:

• To develop a method for producing recombinant mouse Midkine (rmMK) in a prokaryotic system.
• To ensure the quality and bioactivity of the produced rmMK for preclinical use.
• To lay the groundwork for pilot- or large-scale production of rmMK.

Main Methods:

• Mouse Midkine (mMK) open reading frame was cloned into the pET30a (+) expression vector.
• The vector was transformed into Escherichia coli BL21 (DE3) for protein expression.
• Recombinant mouse Midkine (rmMK) was expressed, denatured, renatured, and purified using ion exchange and affinity chromatography.

Main Results:

• Recombinant mouse Midkine (rmMK) was successfully expressed in inclusion bodies and solubilized post-denaturation/renaturation.
• Purified rmMK met the quality and bioactivity requirements for animal studies.
• 13.2 mg of high-quality, bioactive rmMK was obtained from 1 L of LB culture with an 11.4% recovery rate.

Conclusions:

• A robust prokaryotic expression system for producing recombinant mouse Midkine (rmMK) was established.
• The developed method facilitates the production of sufficient quantities of rmMK for preclinical evaluations.
• This work supports the advancement of human Midkine as a potential therapeutic agent for joint protection.