Expression, Purification, Structural and Functional Characterization of Recombinant Human Parvulin 17

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Parvulins are enzymes (peptidyl-prolyl isomerase enzymes, PPIase) that regulate protein folding and function by catalyzing cis-trans isomerization of prolyl bonds.
• Par17 is a hominid-specific parvulin with limited structural and functional characterization, hindering research.

Purpose of the Study:

• To establish optimal conditions for the recombinant expression and purification of active Par17 in E. coli.
• To develop a scalable assay for assessing the PPIase activity of parvulins.

Main Methods:

• Comparative evaluation of E. coli host strains, culture media, inducer concentrations, and incubation conditions for Par17 overexpression.
• Recombinant expression and purification of Par17, Pin1 (a representative parvulin), and their catalytic domains.
• Structural characterization using Size Exclusion Chromatography and Circular Dichroism.
• Development of a homogeneous protease-based fluorimetric assay for PPIase activity.

Main Results:

• Optimized protocols for efficient overexpression and purification of folded, active Par17 and Pin1 proteins.
• Demonstrated the structural integrity and catalytic activity of the purified recombinant proteins.
• Established a scalable fluorimetric assay for measuring parvulin PPIase activity.

Conclusions:

• Successfully developed methods for producing active recombinant Par17, paving the way for detailed structural and functional investigations.
• The developed assay provides a valuable tool for high-throughput screening of parvulin activity.
• These findings represent a significant advancement in the study of parvulins, particularly the under-characterized Par17.