title: Selection of ceramic fluorapatite-binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography.
abstract: Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA-specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N-terminal sequence was found in two selected peptides: F4-2 (KPRSMLH) and F5-4 (KPRSVSG). The peptide F5-4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5-4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage-derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins.
authors.affiliation: Department of Biochemical Engineering, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759, Bremen, Germany.
authors.forename: Tuhidul
authors.initials: T
authors.lastname: Islam
authors.forename: Noor Shad
authors.initials: NS
authors.lastname: Bibi
authors.forename: Rami Reddy
authors.initials: RR
authors.lastname: Vennapusa
authors.forename: Marcelo
authors.initials: M
authors.lastname: Fernandez-Lahore
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D000596:Amino Acids; D001031:Apatites / Q000737:chemistry*; D002021:Buffers; D002516:Ceramics / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D000596:Amino Acids; D001031:Apatites; D002021:Buffers; D019151:Peptide Library; D010455:Peptides; C025105:fluorapatite