Purification Of Synthetic Peptides Research Articles

IgE binding epitope mapping with TL1A tagged peptides

Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis.

Synthetic Peptide Purification via Solid-Phase Extraction with Gradient Elution: A Simple, Economical, Fast, and Efficient Methodology

A methodology was implemented for purifying peptides in one chromatographic run via solid-phase extraction (SPE), reverse phase mode (RP), and gradient elution, obtaining high-purity products with good yields. Crude peptides were analyzed by reverse phase high performance liquid chromatography and a new mathematical model based on its retention time was developed in order to predict the percentage of organic modifier in which the peptide will elute in RP-SPE. This information was used for designing the elution program of each molecule. It was possible to purify peptides with different physicochemical properties, showing that this method is versatile and requires low solvent consumption, making it the least polluting one. Reverse phase-SPE can easily be routinely implemented. It is an alternative to enrich and purified synthetic or natural molecules.

Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography

Protected synthetic peptide intermediates are often hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), usually used for industrial production. It is then challenging to develop alternative chromatographic purification processes. Support-free liquid–liquid chromatographic techniques, including both hydrostatic (centrifugal partition chromatography or CPC) and hydrodynamic (counter-current chromatography or CCC) devices, are mainly involved in phytochemical studies but have also been applied to synthetic peptide purification. In this framework, two new biphasic solvent system compositions covering a wide range of polarity were developed to overcome solubility problems mentioned above. The new systems composed of heptane/tetrahydrofuran/acetonitrile/dimethylsulfoxide/water and heptane/methyl-tetrahydrofuran/N-methylpyrrolidone/water were efficiently used for the CPC purification of a 39-mer protected exenatide (Byetta®) and a 8-mer protected peptide intermediate of bivalirudin (Angiox®) synthesis. Phase compositions of the different biphasic solvent systems were determined by 1H nuclear magnetic resonance. Physico-chemical properties including viscosity, density and interfacial tension of these biphasic systems are also described.

Purification of Synthetic Peptides Using a Catching Full-Length Sequence by Polymerization Approach

During automated solid-phase peptide synthesis, failure sequences were capped with acetic anhydride. After synthesis, a polymerizable methacrylamide tag was attached to the full-length sequences. Peptide purification was then achieved by polymerizing the full-length sequences, washing away impurities, and cleaving the peptide product from the polymer.

ChemInform Abstract: Purification of Synthetic Peptides. Immobilized Metal Ion Affinity Chromatography (IMAC).

ChemInform Abstract: Purification of Synthetic Peptides. Immobilized Metal Ion Affinity Chromatography (IMAC).

Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1 mm to 9.4 mm I.D.)

The present study examines the effect of reversed-phase high-performance liquid chromatography (RP-HPLC) column diameter (1 mm to 9.4 mm I.D.) on the one-step slow gradient preparative purification of a 26-residue synthetic antimicrobial peptide. When taken together, the semi-preparative column (9.4 mm I.D.) provided the highest yields of purified product (an average of 90.7% recovery from hydrophilic and hydrophobic impurities) over a wide range of sample load (0.75–200 mg). Columns with smaller diameters, such as narrowbore columns (150 × 2.1 mm I.D.) and microbore columns (150 × 1.0 mm I.D.), can be employed to purify peptides with reasonable recovery of purified product but the range of the crude peptide that can be applied to the column is limited. In addition, the smaller diameter columns require more extensive fraction analysis to locate the fractions of pure product than the larger diameter column with the same load. Our results show the excellent potential of the one-step slow gradient preparative protocol as a universal method for purification of synthetic peptides.

Structure-Activity Relationships of Neuromedin U. V. Study on the Stability of Porcine Neuromedin U-8 at the C-Terminal Asparagine Amide under Mild Alkaline and Acidic Conditions

Porcine neuromedin U-8 (X-Asn-NH(2), X=H-Tyr-Phe-Leu-Phe-Arg-Pro-Arg) is occasionally unstable in the biological fluids used for bioassay as well as in the acidic solutions used for purification of synthetic peptides. In this study, HPLC examination of an incubate solution of X-Asn-NH(2) revealed that the main decomposition products in Tyrode's solution (pH 7.4) were either alpha- or beta-monocarboxylic acid analogs (X-Asn-OH or X-Asp-NH(2)), and that no dicarboxylic acid analog (X-Asp-OH) was produced. Further investigation, employing a model peptide (Y-Asn-NH(2), Y=Benzoyl-Pro-Arg) incubated in a 0.1 M sodium bicarbonate solution at 60 degrees C, revealed that the decomposition of C-terminal Asn-NH(2) occurred through the formation of an aminosuccinimide intermediate (Y-Asu), at a rate faster than that of Y-Asn-Ser peptide but slower than that of Y-Asn-Gly peptide. Mild acid hydrolysis of X-Asn-NH(2) examined in a 1 M HCl solution at 60 degrees C yielded X-Asn-OH and X-Asp-NH(2), which further decomposed to yield X-Asp-OH. The C-terminal degradation of X-Asn-NH(2) resulted in reduced biological and immunochemical binding activities.

A novel, base-labile fluorous amine protecting group: synthesis and use as a tag in the purification of synthetic peptides

A new, base-labile fluorous tag based on the Msc amine protecting group was synthesized. Its use in the purification of synthetic peptides by fluorous HPLC or fluorous SPE was demonstrated.

Use of benzyloxycarbonyl (Z)-based fluorophilic tagging reagents in the purification of synthetic peptides

Three novel fluorous reagents (i.e. 1– 3 ), derived from benzyl chloroformate (Z-Cl), have been synthesized and used for the tagging of peptides prepared following a Fmoc-based solid-phase approach. It is shown that the implementation of a benzyloxycarbonyl (Z)-based fluorophilic tag facilitates purification of peptides using fluorous reverse phase chromatography.

Reversed-phase poly(styrene–divinylbenzene) materials optimised for large scale preparative and process purification of synthetic peptides and recombinant proteins

Rigid macroporous copolymers of styrene and divinylbenzene have been designed for large-scale preparative and process-scale purification of synthetic peptides and recombinant proteins. The polymeric particles are mechanically stable and hence able to operate at the required high linear velocities. The pore size and pore morphology has been optimised to enable unhindered solute diffusion whilst providing maximum available surface area to enhance loading capacity. A 100 Å pore size has been developed for synthetic peptides and a 300 Å pore size for recombinant proteins. Precise control of particle size, within the range 10 to 20 μm, is possible which together with the very narrow particle size distribution enables maximum resolution/loading to be obtained within the pressure limits of the instrumentation being used. The chemical stability of the polymer enables cleaning in place with 1 M sodium hydroxide without particle dissolution or a deterioration in selectivity. These materials can be packed into compression hardware and are manufactured as single lots up to 100 kg (300 l) batch size.

The Purification of Synthetic Peptides

AbstractFor Abstract see ChemInform Abstract in Full Text.

Hydrophilic interaction/cation-exchange chromatography for the purification of synthetic peptides from closely related impurities: serine side-chain acetylated peptides.

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEC) is a novel HPLC technique which has excellent potential for peptide separations. Separations by HILIC/CEC are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlayed on ionic interactions with the cation-exchange matrix. Complex peptide mixtures produced by solid-phase synthesis are a frequently encountered and challenging purification problem. In the present study a two-step protocol, consisting of HILIC/CEC followed by RPC, was required for the successful purification of a 21-residue synthetic amphipathic alpha-helical peptide from serine side-chain acetylated impurities, with HILIC/CEC proving to be highly sensitive to subtle differences in hydrophilicities between the acetylated peptides and the desired product. Investigation of the three potential sites of serine acetylation through solid-phase synthesis of acetylated analogues of the desired peptide (peptides of the same sequence and secondary structure, but acetylated at different positions on the hydrophilic face of the alpha-helix) demonstrated that acetylation was occurring at different sites on the peptide. HILIC/CEC was able to take advantage of very subtle changes in environment around the acetylation sites and thus effect a separation of these analogues not achievable by RPC or CEC alone.

Synthesis of a versatile purification handle for use with Boc chemistry solid phase peptide synthesis

The synthesis of a versatile handle for the purification of synthetic peptides is described. The Boc-aminoethylsulfonylethyloxycarbonyl handle is coupled to the NH 2-terminus of the resin-bound peptide. Removal of the Boc group affords a free amine which can be derivatized with any of a variety of functionalities. Cleavage from the resin gives a peptide functionalized for covalent chemoselective reaction with the column support. Desired full length peptide is eluted from the column by cleavage of the handle by treatment with base.

Purification of synthetic peptides by high performance liquid chromatography.

Synthesis of peptides on a solid support is described in detail in Chapter 5 of this book. Contributing to the ongoing success of Merrifield’s solid-phase peptide synthesis methodology (1) was the use of high performance liquid chromatography (HPLC) for the purification of the desired peptide from the byproducts generated by this technique.

Purification of synthetic peptides with the aid of reversible chromatographic probes

The application of reversible Chromatographic probes for the purification of synthetic peptides by reversed-phase (RP) and affinity Chromatography is described. In previous work the concept of a one-step purification procedure based on the acid-stable fluorenylmethoxycarbonyl (Fmoc) group was considered. Similar probes were used to purify peptides from 17 to 104 residues in length, using ion-exchange and RP media. The RP probes described here represent simplified versions of those reported previously. Further, the application of a new probe containing biotin, for affinity Chromatography, is demonstrated. Stepwise solid-phase peptide synthesis was performed using both Boc and Fmoc chemistries and N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping reagent. The Chromatographic probes were incorporated on to the “target” peptide as active succinimidyl carbonate derivatives. After cleavage using either hydrogen fluoride or trifluoroacetic acid, purification was performed on either RP media or immobilized avidin. Treatment with organic base yielded the free purified product. The efficiency of the new probe molecules and the capping procedure was demonstrated by the purification of two polypeptides, 46 and 101 residues in length.

ChemInform Abstract: Affinity Purification of Synthetic Peptides and Proteins on Porous Graphitized Carbon.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Peptide purification by reverse-phase HPLC.

AbstractThe increasing sophistication and sensitivity of microsequencing of peptides and proteins in recent years have presented the biochemist with the feasibility of primary structural determinations on low-picomole quantities of material. This has led to a plethora of structural data in many different areas of interest, which would until now have taken decades of effort to produce. Complementary to the increased sensitivity of analytical technology has been the application of HPLC in the rapid and highly discriminating separation of biomolecules. If such fractionations are interfaced with a specific rapid detection system, such as an immunoassay for the peptide or protein of interest, then purification to homogeneity from a crude extract can be accomplished in a matter of days. Purification to homogeneity is an absolute prerequisite for successful and meaningful structural analyses. Reverse-phase HPLC is usually the technique of choice for isolation procedures, but if complex biological material represents the starting point of an isolation, it is wise to perform a degree of sample clean up and/or concentration initially. Much of what applies to peptide isolation from complex biological material is also relevant to purification of synthetic peptides from resin eluates.KeywordsCyanogen BromideAutomate Edman DegradationHeptafluorobutyric AcidColumn EffluentPeptide PurificationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Affinity purification of synthetic peptides and proteins on porous graphitised carbon

The affinity of the Tbfmoc group for porous graphitised carbon has been exploited for the purification of synthetic peptides containing up to 85 amino acids. The hydrophobicity of the group has also been used to simplify peptide purification by HPLC.

Purification of synthetic peptides using reversible chromatographic probes based on the Fmoc molecule.

A rapid, versatile, reversible procedure for purifying synthetic peptides has been developed based on the specific incorporation of 4-carboxylate Fmoc derivatives onto the terminal amino acid of peptidyl-resins. The acid stable 4-COR-Fmoc derivatives were synthesised with a variety of chemical groups thus altering the chromatographic properties of the "target" peptides and permitting their convenient purification, either by reversed-phase HPLC or ion exchange chromatography. The assembly of the peptides involved a capping step to prevent the formation of deletion forms. The 4-COR-Fmoc derivatives were incorporated either as preformed amino acid conjugates or as activated succinimidyl esters. After HF cleavage and purification the 4-COR-Fmoc probes were quantitatively removed with organic bases. The efficiency of the technique was demonstrated by the purification of small to large sized peptides, including a cyclic analogue.

Synthetic Peptide Purification in The Multi-Coil Countercurrent Chromatograph

Abstract The multi-coil countercurrent chromatograph (MC-CCC) is used for preparative separations of various synthetic peptides. For peptides that are water insoluble and that have a high partitioning in the upper phase of n-butanol. acetic acid and water, the use of the more hydro phobic solvent system comprised of chloroform, acetic acid and water provides purification. An alternative two-phase solvent system made up of equal volumes of three components such as ethyl acetate, acetonitrile and an aqueous acid or salt solution is proposed for peptide separation as well.