A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a...

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Recombinant protein production is preferred, but native protein purification persists.
• Native bacteriophage protein isolation is challenging due to large lysate volumes.
• Current methods like ammonium sulfate fractionation are cumbersome and expensive.

Purpose of the Study:

• To develop effective and inexpensive reversible protein precipitation methods.
• To characterize the novel TP-84 bacteriophage TP84_26 protein, a putative capsule depolymerase.
• To establish a scalable purification protocol for bacteriophage proteins.

Main Methods:

• Purification of the 112 kDa TP84_26 protein from infected Geobacillus stearothermophilus 10 cells.
• Confirmation of protein biosynthesis via mass spectrometry (LC-MS) and enzymatic activity assays.
• Development and application of a modified polyethyleneimine (PEI)-mediated purification method.

Main Results:

• The TP84_26 protein, a capsule depolymerase, was successfully purified and characterized.
• Three forms of the depolymerase were identified: soluble, unbound, and virion-integrated.
• Enzymatic activity against G. stearothermophilus polysaccharide capsules was confirmed.

Conclusions:

• The novel TP-84 depolymerase exists in three functional forms.
• Soluble forms likely weaken bacterial capsules, while virion-integrated forms aid phage entry.
• The developed PEI purification method is suitable for industrial-scale bacteriophage protein production.