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

Abstract

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.

MS2 Virus-like Particles as a Versatile Peptide Presentation Platform: Insights into the Deterministic Abilities for Accommodating Heterologous Peptide Lengths

“…Moreover, despite forming inclusion bodies, the expression levels of dMS2-GST and dMS2-eGFP revealed relatively higher levels. This could be explained by the fact that both the GST tag and eGFP are commonly used to promote protein soluble expression. − Efforts to refold the inclusion bodies with 8 M urea yielded undesirable outcomes (data not shown), indicating irreversible damage to the correct folding and assembly of MS2 due to the insertion of excessively long foreign proteins. These results highlighted the critical role of the length and identity of the insertions in determining the efficiency of the MS2 VLPs display, underscoring the importance of carefully selecting appropriate insertions when using the MS2 VLP display platform.…”

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

“…Amphipathic helices of CPC1, FAP297, and FAP266 were affinity-purified using glutathione sepharose beads (Fig. S1; [24]).…”