Surface supercharged human enteropeptidase light chain shows improved solubility and refolding yield

Area of Science:

• Biochemistry
• Protein Engineering
• Biotechnology

Background:

• Enteropeptidase is a serine protease valuable in biotechnology.
• The human enteropeptidase light chain (hEPL) offers high activity but suffers from poor solubility and refolding yields.
• These limitations hinder its broader application in biotechnological processes.

Purpose of the Study:

• To engineer protein modifications for enhanced solubility and refolding of recombinant human enteropeptidase light chain (hEPL).
• To maintain the enzymatic activity of hEPL after protein modifications.
• To improve the stability and yield of hEPL for biotechnological applications.

Main Methods:

• Protein surface supercharging was employed using specific amino acid substitutions (N6D, G21D, G22D, N141D, K209E).
• A free cysteine residue was replaced with serine (C112S) to address refolding issues.
• The impact of these modifications on solubility, refolding yield, and heat stability was evaluated.

Main Results:

• Protein surface supercharging resulted in over a 100-fold increase in hEPL solubility.
• The C112S variant demonstrated a 50% improvement in refolding yield.
• Supercharging further enhanced the heat stability of the C112S variant, indicating improved protein robustness.

Conclusions:

• Protein surface supercharging is an effective strategy to dramatically improve the solubility of human enteropeptidase light chain (hEPL).
• Specific modifications, including cysteine replacement and supercharging, significantly boost refolding yields and stability.
• These engineered hEPL variants show promise for overcoming current limitations in biotechnological applications.