A general platform for efficient extracellular expression and purification of Fab from Escherichia coli

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Antigen-binding fragments (Fabs) are crucial components of monoclonal antibody (mAb) therapeutics.
• Escherichia coli (E. coli) is a cost-effective system for producing Fabs.
• Cytoplasmic expression in E. coli leads to Fab aggregation, reducing yields.

Purpose of the Study:

• To develop a novel system for efficient extracellular production of Fabs in E. coli.
• To overcome Fab aggregation issues in the E. coli cytoplasm.
• To evaluate the efficacy of a new system using the alkaline phosphatase (phoA) promoter and heat-stable enterotoxin II (STII) leader sequence.

Main Methods:

• Utilized five different Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1.
• Employed the alkaline phosphatase (phoA) promoter and heat-stable enterotoxin II (STII) leader sequence for expression and secretion.
• Expressed Fabs in BL21(DE3) E. coli strain under phosphate starvation conditions.
• Purified secreted Fabs using affinity chromatography specific to the constant region of the light chain.

Main Results:

• All five Fab fragments were efficiently expressed and secreted into the culture medium.
• Purified Fabs demonstrated high purity, antigen-binding affinity, and in vitro bioactivity.
• The BL21(DE3) strain showed higher productivity than K-12 strains.
• The phoA promoter exhibited superior secretion ability compared to the T7 promoter.
• The STII signal peptide demonstrated higher extracellular secretion efficiency than pelB.

Conclusions:

• The developed phoA-STII system facilitates efficient extracellular production of Fabs in E. coli.
• This platform overcomes aggregation issues, enhancing yield and purity of functional Fabs.
• The system holds significant promise for the manufacturing of Fab fragments in academic and industrial settings.