An efficient depyrogenation method for recombinant bacterial outer membrane lipoproteins

Area of Science:

• Microbiology
• Immunology
• Biochemistry

Background:

• Bacterial outer membrane lipoproteins are difficult to purify due to their association with lipopolysaccharides (LPS) and hydrophobic proteins.
• Previous work highlighted delipidation as crucial for purifying recombinant antigens fused to Pseudomonas aeruginosa OprI lipoprotein.

Purpose of the Study:

• To report cloning and expression of three antigens fused to OprI.
• To address challenges in reducing variable lipopolysaccharide (LPS) contamination in purified lipoproteins.
• To develop a scalable method for purifying recombinant lipoproteins with low LPS content.

Main Methods:

• Cloning and expression of ovalbumin, eGFP, and BbPDI antigens fused to OprI.
• Evaluation of polymyxin B columns, endotoxin removal beads, Triton X-114, and sodium deoxycholate for LPS removal.
• Implementation of a hot phenol/water LPS extraction prior to metal affinity chromatography.

Main Results:

• Polymyxin B and magnetic beads caused high protein loss; detergents were ineffective for LPS reduction.
• Hot phenol/water extraction followed by metal affinity chromatography yielded purified lipoproteins with LPS levels below 0.02 EU/μg.
• Purified recombinant lipoproteins maintained their ability to stimulate bone marrow-derived dendritic cells via TLR2/1.

Conclusions:

• Hot phenol/water extraction is an effective and scalable method for purifying bacterial outer membrane lipoproteins with low LPS contamination.
• This method facilitates the study of immunomodulatory properties of recombinant lipoproteins.
• The technique is applicable to the purification of other outer membrane lipoproteins.