Production of a recombinant, 40 amino acid helix-loop-helix peptide (EF2-GGC) for incorporation into a novel affinity purification system

Fragment complementation between EF1 and EF2, calcium binding protein subdomains of the protein calbindin D9k, forms the basis behind a novel affinity protein purification system enabling rapid, highly specific capture and elution of pure EF1-tagged protein in physiological conditions. Construction of an affinity resin for this purification system relies on efficient production of the EF2 peptide as the affinity ligand to be displayed on a nanoscale scaffold.In this thesis a protocol was developed for production of the EF2 peptide via a recombinant fusion protein construct incorporating the EF2 peptide with an added terminal GGC. Isolation of pure EF2-GGC peptide from this fusion protein followed. This protocol involved expression of this recombinant protein through the bacterial cell host, Escherichia coli (E.coli), and purification via native cell lysis and immobilized metal affinity chromatography (IMAC), both optimized for yield and purity. Subsequent processing steps of this recombinant fusion protein consisted of TEV protease cleavage and additional affinity purification steps to obtain the peptide in a pure formulation for further steps in affinity resin manufacture.Initial protein expression investigations demonstrated that the EF2-GGC recombinant fusion protein was soluble and expressed well in both autoinduction and isopropyl β-D-1-thiogalactopyranoside (IPTG) induction systems. Ni-NTA IMAC purification of this recombinant fusion protein was successful with a yield of approximately ~50 mg/L. Proteolytic cleavage of the EF2-GGC recombinant fusion protein successfully releasing the EF2-GGC peptide of interest was optimised. A second round of Ni-NTA IMAC, ion exchange chromatography (IEX) and a final polishing step of size exclusion chromatography (SEC) retrieved the peptide in a highly pure form, suitable for subsequent resin coupling steps.While this protocol was optimised to provide 6 - 12 mg/L of recombinant EF2-GGC peptide, SEC analysis confirmed that the peptide was prone to significant aggregation. Further investigations into the peptide buffer formulation failed to completely reverse aggregation. Therefore, while the expression and purification protocols developed during this project have been effective in isolating a pure EF2-GGC peptide, further work to identify an effective formulation for the purified peptide is required