Selection for a periplasmic factor improving phage display and functional periplasmic expression

Area of Science:

• Molecular Biology
• Biotechnology
• Protein Engineering

Background:

• Periplasmic folding is a critical step affecting the efficiency of phage display and recombinant protein expression in Escherichia coli.
• Identifying factors that improve these processes is crucial for advancing biotechnological applications.

Purpose of the Study:

• To discover Escherichia coli (E. coli) factors that enhance both phage display and periplasmic expression of recombinant proteins.
• To investigate the role of periplasmic proteins in improving the display of poorly folding antibody fragments.

Main Methods:

• A library of E. coli proteins was coexpressed with a poorly folding single-chain variable fragment (scFv) antibody fused to g3p in a phagemid vector.
• Panning for antigen binding was used to enrich phagemids with higher amounts of displayed scFv.
• The effect of coexpressed periplasmic proteins on scFv display and functional yield was analyzed.

Main Results:

• The periplasmic protein Skp (also known as OmpH/HlpA) was identified as a factor that significantly improves phage display of various scFv fragments.
• Coexpression of Skp increased the amount of hybrid protein displayed on the phage.
• Coexpression of skp also led to a higher functional yield of scFv fragments secreted to the periplasm.

Conclusions:

• Skp is a key E. coli protein that enhances the efficiency of phage display by increasing the displayed amount of scFv fragments.
• Skp also improves the functional yield of recombinant scFv fragments expressed via periplasmic secretion.
• This finding has implications for improving antibody engineering and recombinant protein production platforms.