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.

Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality

“…To overcome these limitations, a number of small synthetic ligands for IgG have been developed, including triazine scaffolds, nucleic acids, multimodal ligands, chelated metals, single amino acids, and peptides [14,15]. The latter class in particular—the peptide ligands—shows considerable promise as alternative to protein ligands [16,17,18]. Short peptides are generally nontoxic, chemically stable, and can be produced synthetically, hence relatively inexpensively, on the large scale, and with no batch-to-batch variability [19,20].…”

A modular approach to multifunctional polypeptide/ceramic fluorapatite‐based self‐assembled system in affinity chromatography for the purification of human Immunoglobulin G

“…Therefore, the mobile phase, which will be used for the reversible interactions between hIgG and the ligand, should have the ability to prohibit the retention of biomolecules on the CFT matrix. It has been observed in our previous study that the retention of biomolecules on CFT can be fine‐tuned by the composition of Na‐phosphate buffer (Islam et al ., ). Therefore, in this study, 0 to 200 mM NaCl in 10, 20, or 30 mM Na‐phosphate was used to optimize the buffer's composition to restrict the interactions between biomolecules and CFT at pH 7.4 and 6.…”

“…Recently, Yang et al have identified a hexamer peptide sequence HWRGWV (pLI) that can bind with a high affinity and specificity to hIgG (Yang et al ., ; Yang et al ., ). On the other hand, CFT‐binding heptamer peptides were identified using the phage display selection (Islam et al ., ). The preferential affinity and specific retention properties showed that the CFT‐binding peptide sequence KPRSVSG (pTC) can be used for the physical immobilization of a ligand on CFT.…”