mRNA display selection and solid‐phase synthesis of Fc‐binding cyclic peptide affinity ligands

Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid‐phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on‐resin solid‐phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide—cyclo[Link‐M‐WFRHY‐K]—as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link‐M‐WFRHY‐K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link‐M‐WFRHY‐K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link‐M‐WFRHY‐K] is an attractive candidate for developing a cost‐effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest. Biotechnol. Bioeng. 2013; 110: 857–870. © 2012 Wiley Periodicals, Inc.     

Monolithic peptidyl sorbents for comparison of affinity properties of plasminogen activators

Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein's denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA-EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.     

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

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. Copyright © 2013 John Wiley & Sons, Ltd.     

Rational design of peptide affinity ligands for the purification of therapeutic enzymes

Non‐mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof‐of‐concept for developing affinity peptide‐based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:987–998, 2018     

Comparative studies of adhesion peptides based on l‐ or d‐amino acids

Detailed studies comparing solid‐supported l ‐ or d ‐amino acid adhesion peptides based on the sequence KLHRIRA were performed. Stability towards proteases and levels of cellular adhesion to the otherwise inert surface of PEGA resin were compared by using fluorescently labelled peptides. A clear difference in the peptide stability towards cleavage by subtilisin, trypsin, or papain was observed. However, all of the on‐bead peptides provided an optimal surface for cell adhesion and proliferation. In long‐term experiments, these properties were still found to be similar on the resins modified either with l ‐ or with d‐ amino acids and unaffected by the nature of their fluorescence labelling at either terminus. These results support that the more accessible l ‐amino acids can be utilized for cell adhesion experiments and confirm the nonspecific interaction mechanism of cell binding to these peptides on the bead surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Solid phase peptide synthesis on epoxy-bearing methacrylate monoliths.

Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing beta-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.     

Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA)

Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.     

Hydroxyapatite affinity chromatography for the highly selective enrichment of mono‐ and multi‐phosphorylated peptides in phosphoproteome analysis

The most challenging analytical task facing phosphoproteome determination requires the isolation of phosphorylated peptides from the myriad of unphosphorylated species. In the past, several strategies for phosphopeptide isolation have been proposed in combination with subsequent mass spectrometric investigations. Among these techniques, immobilized metal affinity chromatography and titanium dioxide have been recognized as the most effective. Here, we present an alternative method for the enrichment of phosphopeptides based on hydroxyapatite (HAP) chromatography. By taking advantage of the strong interaction of HAP with phosphate and calcium ions, we developed an efficient method for the selective separation and fractionation of phosphorylated peptides. The effectiveness and efficiency of recovery for this procedure was assayed using tryptic digests of standard phosphorylated protein mixtures. Based on the higher affinity of multi‐phosphorylated peptides for HAP surfaces, the introduction of a phosphate buffer gradient for stepwise peptide elution resulted in the separation of mono‐, di‐, tri‐, and multi‐phosphorylated peptides. Thus, we demonstrated that this technique is highly selective and independent of the degree of peptide phosphorylation.     

Production of recombinant peptides as fusions with SUMO

Recombinant production of non-native peptides requires using protein fusion technology to prevent peptide degradation by host-cell proteases. In this work, we have used SUMO protein as a fusion partner for the production of difficult-to-express, antimicrobial, self-assembling and amyloidogenic peptides using Escherichia coli. SUMO-peptide fusions were expressed as intracellular products by utilizing pET based expression vectors constructed by Life Sensors Inc., USA. Histidine tagged SUMO-peptide fusions were purified using Ni-NTA affinity chromatography. Complete (100%) cleavage of the SUMO-peptide fusion was achieved using SUMO protease-1. Our findings demonstrate that SUMO fusion technology is a promising alternative for production of peptides in E. coli. The key advantage of this technology is that the enzymatic activity of SUMO protease-1 is specific and efficient leading to inexpensive costs for cleaving the peptide fusion when compared with other fusion systems.     

Solution stability of linear vs. cyclic RGD peptides.

Arg-Gly-Asp (RGD) peptides contain an aspartic acid residue that is highly susceptible to chemical degradation and leads to the loss of biological activity. Our hypothesis is that cyclization of RGD peptides via disulphide bond linkage can induce structural rigidity, thereby preventing degradation mediated by the aspartic acid residue. In this paper, we compared the solution stability of a linear peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2) as a function of pH and buffer concentration. The decomposition of both peptides was studied in buffers ranging from pH 2-12 at 50 degrees C. Reversed-phase HPLC was used as the main tool in determining the degradation rates and pathways of both peptides. Fast atom bombardment mass spectrometry (FAB-MS), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) were used to characterize peptides 1 and 2 and their degradation products. In addition, co-elution with authentic samples was used to identify degradation products. Both peptides displayed pseudo-first-order kinetics at all pH values studied. The cyclic peptide 2 appeared to be 30-fold more stable than the linear peptide 1 at pH 7. The degradation mechanisms of linear (1) and cyclic (2) peptides primarily involved the aspartic acid residue. However, above pH 8 the stability of the cyclic peptide decreased dramatically due to disulphide bond degradation. Both peptides also exhibited a change in degradation mechanism upon an increase in pH. The increase in stability of cyclic peptide 2 compared to linear peptide 1, especially at neutral pH, may be due to decreased structural flexibility imposed by the ring. This rigidity would prevent the Asp side chain carboxylic acid from orientating itself in the appropriate position for attack on the peptide backbone.     

Identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of phage display.

A proof-of-principle study was initiated to determine whether phage-display technology could be used to identify peptides as leads in the customization of ligands for affinity chromatography and to identify a peptide or peptidomimetic for use as a Protein A alternative in the affinity purification of monoclonal antibodies. The constant region of humanized anti-Tac (HAT), prepared by pepsin digestion and receptor-affinity chromatography, was used as the target for phage display in this study. As such, 20 phage-derived peptide sequences were identified from four rounds of biopanning with two linear phage-display libraries (7-mer, containing 100 copies of 2 x 10(9) sequences and 12-mer, containing 70 copies of 1.4 x 10(9) sequences). Five peptides were synthesized for use as affinity ligands, based on sequence homology to Protein A, sequence redundancy, and amino acid motifs. The best HAT binding immobilized peptide was EPIHRSTLTALL. The best-fit analysis of this peptide sequence with Protein A yielded an alignment well within the Fc binding domain of Protein A. These results suggest that phage display can serve as a tool in the identification of peptides as model ligands for affinity chromatography.     

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix‐assisted laser desorption/ionisation mass spectrometry

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of ‘one‐bead‐one‐peptide’ combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4‐hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc‐Asp[2‐phenylisopropyl (OPp)]‐OH to Ala‐Gly‐oxymethylbenzamide‐ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N‐terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N‐Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one‐bead‐one‐cyclic depsipeptide libraries that can be easily open for its sequencing by matrix‐assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Preparation,isolation and identification of iron-chelating peptides derived from Alaska pollock skin

Alaska pollock skin as aquatic by-product was usually discarded in food processing. Iron deficiency has been a nutritional problem for a long period. In this study, Alaska pollock skin was used to generate iron-chelating peptides after being treated by commercial enzymes. Sequential chromatography, including immobilized metal affinity chromatography (IMAC) and reversed phase high performance liquid chromatography (RP-HPLC), was used for capturing and purifying iron-chelating peptides. One tripeptide, with high iron-chelating activity, was obtained and identified by electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). The amino acid sequence of the iron-chelating peptide was identified to be Ser-Cys-His (MW: 345 Da). These results show that Alaska pollock skin could be utilized to generate iron-chelating peptides as an iron supplement in functional food industry.     

Recombinant protein purification using complementary peptides as affinity tags

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.     

Improvement of biological activity and proteolytic stability of peptides by coupling with a cyclic peptide

The cyclic moiety of an endothelin antagonist peptide RES-701-1, composed of 10 amino acids with an amide bond between alpha-NH(2) of Gly1 and beta-COOH of Asp9, was coupled to some biologically active peptides aiming to improve their activities and stabilities against proteolytic degradation. Coupling of the cyclic peptide to the N-terminal of RGD-peptides, maximally 4-fold improvement of in vitro activity compared to the original peptide has been achieved. Coupling of it to protein farnesyltransferase inhibiting peptides resulted to improve in vitro activity maximally 3-fold. These peptides coupled with the cyclic peptide also showed enhanced stability against some typical proteases. These results indicate that this cyclic peptide can stabilize the conformations of the peptides coupled to its C-terminus. Coupling of our cyclic peptide is anticipated to be a novel conformational stabilizing method for biologically active peptides, results to improve their activity and stability.     

Antibody-Defined Epitope Mapping Using the Multipin Method of Peptide Synthesis

The multipin method for peptide synthesis was developed for antibody epitope mapping. From its initial invention as a method for testing many short peptides on the solid surface used for their synthesis, it has evolved into a method for producing solutions of peptides of a wide range of lengths in many “formats,” such as tagged (biotinylated) peptides for binding studies or affinity chromatography. The power of the multipin technique lies in the large number and variety of peptides that can be rapidly made and efficiently tested.     

Synthesis,characterization, and biological activity of poly(arginine)‐derived cancer‐targeting peptides in HepG2 liver cancer cells

The solid‐phase synthesis, structural characterization, and biological evaluation of a small library of cancer‐targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose‐regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3–12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14–46%) and crude purities >95% as analyzed by liquid chromatography–mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure–activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose‐regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose‐response (0–1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer‐targeting delivery agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Purification of soluble and membrane-bound proteases with substrate-analogous inhibitors by affinity chromatography.

Specific modified substrate-analogous amino acids and peptides have been used as affinity ligands in the affinity chromatography of proteases. Alanine methyl ketone-Sepharose (AMK-Sepharose) is introduced as affinity support for the purification of a bacterial alanyl aminopeptidase (AAP) from a membrane protein extract and Arginine-Agarose as support for the preparation of a membrane-bound proteinase of myeloma cells (MP-1). Peptidyl methyl ketones as affinity ligands have been used to separate subtilisin enzymes and the cysteine proteases cathepsin B, L, and S. As a new type of ligands, spacer-bound peptidyl chloromethyl ketones are presented for a specific and oriented immobilization of proteinases. Oriented-immobilized cathepsin B was used to isolate antibodies against this enzyme.     

Selection of affinity ligands for protein purification from proteolytic digests

A simple method for the selection of affinity ligands from proteolytic digests by affinity chromatography is proposed. A small proportion of the peptides in the trypsin digest of bovine serum albumin (BSA) or the pepsin digest of cytochrome are retarded on insulin-immobilised or HSA (human serum albumin)-immobilised affinity columns, respectively. The peptides in these selected fractions can be immobilised onto solid phases and used in affinity chromatography procedures for the purification of insulin or HSA. © Rapid Science Ltd. 1998     

Quantum dots laser desorption/ionization MS: multifunctional CdSe quantum dots as the matrix,concentrating probes and acceleration for microwave enzymatic digestion for peptide analysis and high resolution detection of proteins in a linear MALDI‐TOF MS

We report the first use of functionalized cadmium selinide quantum dots (CdSe QDs) with 11‐mercaptoundecanoic acid (MUA) as the matrix for the selective ionization of proteins with high resolution and rapid analysis of amino acids and peptides by using quantum dots laser desorption/ionization mass spectrometry (QDLDI‐MS). The mercaptocarboxylic groups of CdSe QDs have been known to be an effective affinity probe to interact with the biomolecules at low abundance level. Using these QDs as the matrix, sensitivity of the method was greatly enhanced and the LOQ of peptides was found to be 100 pM with RSD <10%. The QDLDI‐MS is capable for the selective ionization of insulin, lysozyme and myoglobin with high resolution, which is not observed with sinapic acid (SA) as the matrix. The QDLDI‐MS technique offers many advantages for the analysis of amino acids, peptides and proteins with regard to simplicity, rapidity, sensitivity and the mass spectra were generated in the presence of signal suppressors such as urea and Trition X‐100. In addition, the CdSe QDs have been successfully applied as preconcentrating probes for the analysis of digested peptides in lysozyme from chicken egg white by microwave‐assisted enzymatic digestion. This indicates that the QDs are able to absorb radiation from microwave and their ability to trap peptides from microwave‐digested lysozyme. These results demonstrate that the CdSe QDs are promising candidates for the selective ionization of the analytes with an accurate platform to the rapid screening of biomolecules.