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What is Peptide Purification?

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  • Written By: Helga George
  • Edited By: Michelle Arevalo
  • Images By: Matt Janicki, Photographee.eu, Alexander Raths
  • Last Modified Date: 01 November 2016
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Peptides are small polymers composed of amino acids. Many are biologically-active as hormones, toxins, or have other abilities. Such compounds are frequently used in biological, medical, and chemical research. They also serve as the building blocks of proteins when numbers of them are linked together. Peptide purification is a technique used to either purify large quantities of a desired peptide, or to help separate peptides generated from the digestion of proteins.

The purification of peptides is accomplished by using a technique of chromatography, a way of separating compounds that bind differentially to a physical matrix. High pressure liquid chromatography (HPLC) is commonly used for peptide purification. The peptide or peptides are applied in a mixture of solvents that changes over time as they are pumped across a column of small beads at high pressure. Different peptides elute at varying time points and are detected by a monitor, frequently a UV spectrophotometer.

When conducting research on peptides, often the substance of interest is rare and cannot be purchased from chemical companies. If the structure of the desired peptide is known, it is frequently easier to prepare chemically by peptide synthesis than to isolate the compound from natural sources. Natural products isolation is notoriously difficult. Synthetic peptides are generally purified by HPLC.

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Such chemical synthesis may be daunting for an independent researcher who is not a chemist. Frequently, this task is contracted out to peptide synthesis companies that specialize in these techniques. This may be more economical than trying to set up the system from scratch in a laboratory. Peptide companies can make custom peptides tailored to the needs of the researcher.

Another reason for peptide purification can be when a researcher has purified a protein and is trying to determine its identity. He or she can degrade the protein into peptide fragments, separate the fragments by purification, and have the fragmentation pattern of the peptides detected by a mass spectrometer as they elute from the column. This technique is known as LC-MS, short for liquid chromatography-mass spectrometry. It gives the molecular weight and amino acid composition of the fragments, and often enables the identification of proteins, if similar or identical ones have been identified previously.

Many researchers work with peptides that have novel features, such as unnatural amino acids, to try and find ones with unusual biological activities. There is a whole field called peptidomimetics devoted to the study of such novel peptides. Often, computer-generated sequences are designed, and a peptide library is synthesized that encompasses a range of atypical peptides. Peptide purification is used to separate individual members of this library to provide pure peptide to test for biological activity. This strategy has been successful in generating at least one new commercially-available drug.

Many of the biologically-active peptides are of interest for medical uses. Compounds used commercially, such as insulin, are commonly produced by recombinant DNA overexpression systems that generate large amounts of the desired compound. Frequently, the peptides are genetically engineered to facilitate purification by having some sort of tag added to their fronts. This allows the use of affinity chromatography to purify the peptide.

With this type of chromatography, the tag is a compound, such as histidine, that will bind a matrix of beads, like nickel, chosen for its ability to bind the tag. Unwanted proteins and peptides generally pass through the column without binding. The specifically designed peptide usually binds strongly to the column. After the contaminating proteins and peptides have been washed off, the desired peptide is eluted by a compound that competes for binding to the matrix. One then has a fairly pure preparation of the desired peptide, and the tag can be removed by cleavage.

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