Improved Production and Biophysical Analysis of Recombinant Silicatein-α

Area of Science:

• Biochemistry
• Enzymology
• Biotechnology

Background:

• Silicatein-α, a hydrolase from siliceous sponges, catalyzes silicon-oxygen bond reactions, offering potential for sustainable siloxane synthesis.
• Previous research on silicatein-α was limited by protein aggregation and insolubility issues.
• The enzyme's unique mechanism and potential applications in materials science warrant further investigation.

Purpose of the Study:

• To develop an improved method for producing a soluble and stable silicatein-α fusion protein.
• To compare the yield, stability, and catalytic activity of silicatein-α with different purification tags.
• To investigate the influence of purification tags on the enzyme's native catalytic properties.

Main Methods:

• Engineered a trigger factor-silicatein fusion protein utilizing a Strep-II tag, replacing the previous hexahistidine tag.
• Assessed protein yield, solubility, and colloidal stability using light scattering and thermal denaturation analyses.
• Evaluated the enzyme's catalytic activity through enzymatic assays.

Main Results:

• Achieved a 244-fold improvement in protein yield with the Strep-II tagged fusion protein compared to previous methods.
• Strep-tagged silicatein-α exhibited enhanced colloidal stability and solubility, although some oligomerization persisted.
• The Strep-tagged protein retained catalytic competency but displayed lower activity than the His-tagged version, suggesting non-specific catalysis by the hexahistidine tag.

Conclusions:

• The Strep-II tag facilitates higher yield and improved stability of silicatein-α fusion proteins.
• Hexahistidine tags may contribute non-specific catalytic activity, impacting the assessment of native enzyme function.
• This work provides a more stable and high-yield source of silicatein-α for studying its intrinsic enzymatic properties and applications.