Recombinant expression, purification and initial characterization of human BBS2+9

Abstract

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder in which a single mutation in one of the BBS genes leads to exceptional clinical heterogeneity. A subset of the BBS genes express proteins that assemble an octameric complex known as the BBSome complex. The BBSome complex consists of seven BBS proteins (BBS1, 2, 4, 5, 7, 8 and 9) and BBIP10. The complex and its subunits are known to be essential for biosynthesis, maintenance and signaling of the cilia. The BBS-chaperonin complex is known to stabilize BBS7 which interacts with BBS2 and BBS9 to form the BBSome core complex. Studies on native bovine BBSome show that BBS2, 7 and 9 share an identical organization of domains. Although more details have been emerging of the structure and function of the subunits, little is known about the oligomeric state, interaction network of the subunits and the pathway of assembly of the complex. Thus, this thesis was aimed at recombinant production and biophysical characterization of the subunits constituting the core complex. Two of the subunits of the core complex, BBS2 and BBS9 were co-expressed in insect cells in soluble form and purified using a combination of different chromatographic methods. Initial biophysical characterization of the BBS2+9 reveals that the protein complex forms an oligomer of higher order or is part of an aggregate. Analysis by Negative Staining Electron Microscopy suggests the purified complex possibly assembles into a V-shaped particle.

Date of Award	2022
Original language	English
Awarding Institution	Vrije Universiteit Brussel