Recombinant Glargine Insulin Production Process Using Escherichia coli

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Diabetes mellitus requires effective blood glucose management.
• Long-acting insulin analogs like glargine insulin are crucial for glycemic control.
• Efficient and scalable production of insulin analogs is essential.

Purpose of the Study:

• To establish a robust production process for high-purity recombinant human glargine insulin.
• To develop a method for blocking the formation of Arg (B31)-insulin.
• To create a model process applicable to other human insulin analogs.

Main Methods:

• Construction of the pPT-GI vector for expressing prepeptide glargine insulin in Escherichia coli JM109.
• Fed-batch fermentation of transformed E. coli, achieving a final dry cell mass of 18 g/l.
• Refolding of expressed inclusion bodies, citraconylation, and trypsin cleavage for enzymatic conversion.
• Purification using ion-exchange and reverse-phase chromatography.
• Analysis by High-performance liquid chromatography (HPLC) and Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

Main Results:

• Prepeptide glargine insulin constituted 38.52% of total protein in E. coli.
• Citraconic anhydride treatment increased enzymatic conversion yield by 3.2-fold.
• Recombinant human glargine insulin was obtained with 98.11% purity.
• The produced insulin analog was verified to be equivalent to the standard human glargine insulin.

Conclusions:

• A high-purity recombinant human glargine insulin production process was successfully established.
• A method to effectively block Arg (B31)-insulin formation was developed.
• The demonstrated process serves as a potential model for producing other human insulin analogs.