A-kinase anchoring proteins are enriched in the central pair microtubules of motile cilia in Chlamydomonas reinhardtii.

Cilia are microtubule-based sensory organelles present in a number of eukaryotic cells. Mutations in the genes encoding ciliary proteins cause ciliopathies in humans. A-kinase anchoring proteins (AKAPs) tether ciliary signaling proteins such as protein kinase A (PKA). The dimerization and docking domain (D/D) on the RIIα subunit of PKA interacts with AKAPs. Here, we show that AKAP240 from the central-pair microtubules of Chlamydomonas reinhardtii cilia uses two C-terminal amphipathic helices to bind to its partner FAP174, an RIIα-like protein with a D/D domain at the N-terminus. Co-immunoprecipitation using anti-FAP174 antibody with an enriched central-pair microtubule fraction isolated seven interactors whose mass spectrometry analysis revealed proteins from the C2a (FAP65, FAP70, and FAP147) and C1b (CPC1, HSP70A, and FAP42) microtubule projections and FAP75, a protein whose sub-ciliary localization is unknown. Using RII D/D and FAP174 as baits, we identified two additional AKAPs (CPC1 and FAP297) in the central-pair microtubules.

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli.

Expression of affinity-tagged recombinant proteins for crystallography, protein-protein interaction, antibody generation, therapeutic applications, etc. mandates the generation of high-yield soluble proteins. Although recent developments suggest the use of yeast, insect, and mammalian cell lines as protein expression platforms, Escherichia coli is still the most popular, due mainly to its ease of growth, feasibility in genetic manipulation and economy. However, some proteins have a spontaneous tendency to form inclusion bodies (IBs) when over-expressed in bacterial expression systems such as E. coli, thus posing a challenge in purification and yield. At times, small peptides undergo degradation during protein production and hence using suitable tags could circumvent the problem. Although several independent techniques have been used to solubilize IBs, these cannot always be applied in a generic sense. Although tagging a GST moiety is known to enhance the solubility of fusion proteins in E. coli, resulting in yields of 10-50 mg/L of the culture, the inherent nature of the protein sequence at times could lead to the formation of IBs. We have been working on a Myc Binding Protein-1 orthologue, viz. Flagellar Associated Protein 174 (FAP174) from the axoneme of Chlamydomonas reinhardtii that binds to an A-Kinase Anchoring Protein 240 (AKAP240) which has been annotated as Flagellar Associated Protein 65 (FAP65). Using an in-silico approach, we have identified two amphipathic helices on FAP65 (CrFAP65AH1 and CrFAP65AH2) that are predicted to bind to FAP174. To test this prediction, we have cloned the GST-tagged peptides, and overexpressed them in E. coli that have resulted in insoluble IBs. The yields of these over-expressed recombinant proteins dropped considerably due to IB formation, indicating aggregation. An integrated approach has been used to solubilize four highly hydrophobic polypeptides, viz. two amphipathic helices and the respective proline variants of FAP65. For solubilizing these polypeptides, variables such as non-denaturing detergents (IGEPAL CA-630), changing the ionic strength of the cell lysis and solubilization buffer, addition of BugBuster®, diluting the cell lysate and sonication were introduced. Our statistically viable results yielded highly soluble and functional polypeptides, indiscreet secondary structures, and a yield of ~ 20 mg/L of the E. coli culture. Our combinatorial strategy using chemical and physical methods to solubilize IBs could prove useful for hydrophobic peptides and proteins with amphipathic helices.