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High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution
A G Tomasselli1, D J Paddock, T L Emmons
1Global Research and Development, St. Louis Laboratories, Pfizer Inc., Chesterfield, MO 63017, USA. alfredo.tomasselli@pfizer.com
Protein and Peptide Letters
|February 22, 2008
Summary
Researchers developed and purified active beta-secretase (BACE) enzyme constructs for structural studies. These BACE enzyme preparations enabled the formation of inhibitor complexes and yielded high-resolution crystals for analysis.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Beta-site APP cleaving enzyme (BACE) is a key aspartyl protease involved in amyloid precursor protein (APP) processing.
- BACE activity is implicated in the generation of the Abeta peptide, a component of amyloid plaques in Alzheimer's disease.
- Understanding BACE structure and function is crucial for developing therapeutic inhibitors.
Purpose of the Study:
- To develop and characterize novel expression constructs for active beta-secretase (BACE).
- To establish a scalable purification protocol for obtaining homogeneous and active BACE enzyme.
- To facilitate structural studies of BACE, including complex formation with inhibitors.
Main Methods:
- Over-expression of three distinct BACE constructs (pET11a-BACE, pQE80L-BACE, pQE70-BACE) in Escherichia coli.
- Solubilization of inclusion bodies in urea and subsequent refolding by dilution to yield active enzyme.
- Purification of active BACE using anion-exchange and affinity chromatography.
Main Results:
- Maximal activity for pET11a-BACE and pQE80L-BACE was achieved within 3-4 days, while pQE70-BACE required approximately 21 days.
- A scalable purification process yielded over 400 mg of active BACE enzyme.
- Purified BACE constructs formed complexes with inhibitors, and pQE70-BACE yielded crystals diffracting up to 1.45 A resolution.
- A ligand-free crystal form of pQE70-BACE was obtained, useful for preparing inhibitor complexes.
Conclusions:
- The developed BACE expression and purification methods provide a scalable source of active enzyme for structural biology.
- High-resolution crystal structures of BACE, including inhibitor complexes and a ligand-free form, were obtained.
- These findings are instrumental for structure-based drug design targeting BACE inhibition.
