Expression and purification of full-length human Bax alpha

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Bax is a proapoptotic protein of the Bcl-2 family, implicated in mitochondrial membrane potential disruption during apoptosis.
• Previous studies were limited to C-terminally truncated Bax due to the protein's hydrophobic C-terminus and poor solubility.
• Recombinant full-length Bax has not been extensively studied previously.

Purpose of the Study:

• To express and purify full-length human Bax alpha.
• To characterize the ion channel forming activity of full-length Bax.
• To compare the functional efficiency of full-length Bax with its C-terminally truncated counterpart.

Main Methods:

• Expression of full-length human Bax alpha with an N-terminal His tag.
• Purification using Ni-NTA-agarose affinity chromatography and Q-Sepharose ion-exchange chromatography.
• Assessment of protein purity by SDS-PAGE and functional assay using liposomes and carboxyfluorescein release.

Main Results:

• Successfully expressed and purified highly pure (>98%) full-length human Bax alpha.
• The purified protein was soluble and could be concentrated in the presence of octyl glucoside.
• Full-length Bax exhibited 25-fold greater efficiency in forming ion channels and triggering carboxyfluorescein release from liposomes compared to C-terminal truncated Bax.

Conclusions:

• Full-length Bax can be successfully expressed, purified, and functionally characterized.
• The C-terminus is critical for Bax's ion channel activity and proapoptotic function.
• These findings provide a foundation for further structural and functional studies of full-length Bax.