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In this work, we employed fusions of affinity peptides and elastin-like polypeptide (ELP) to carry out a proof-of-concept study for the single-step purification of model, tag-free proteins. Three known peptide-protein binding pairs of varied binding strengths were evaluated. The peptide-protein binding was first characterized through in-solution binding techniques such as fluorescence spectroscopy. Peptide-ELP constructs were then produced in E. coli and purified by phase transition. The binding of the peptide-ELP constructs to the products were then evaluated using competitive fluorescence spectroscopy. Affinity capture, precipitation and recovery experiments were then conducted using the three constructs to evaluate the efficacy of these peptide-ELP based affinity precipitation processes. Two out of the three systems tested were successful in capturing the product and yielding pure protein in a single affinity precipitation step. These results indicated that peptide-protein affinity played an important role in both the effective capture of the protein, and also in the required binding molar ratio for the affinity precipitation process. In addition, for intermediate affinity systems, constructs containing two copies of the peptide at the N-terminus of the ELP were beneficial towards achieving both high purity and yield, likely due to increased affinity from avidity effects. This proof-of-concept study lays the foundations for the development of new peptide-ELP-based affinity purification processes for industrially relevant proteins and other classes of biologics.
This article was published in the following journal.
Name: Journal of biotechnology
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Cell surface proteins that bind acetylcholine with high affinity and trigger intracellular changes influencing the behavior of cells. Cholinergic receptors are divided into two major classes, muscarinic and nicotinic, based originally on their affinity for nicotine and muscarine. Each group is further subdivided based on pharmacology, location, mode of action, and/or molecular biology.
Cell surface proteins that bind VASOACTIVE INTESTINAL PEPTIDE; (VIP); with high affinity and trigger intracellular changes which influence the behavior of cells.
The production of PEPTIDES or PROTEINS by the constituents of a living organism. The biosynthesis of proteins on RIBOSOMES following an RNA template is termed translation (TRANSLATION, GENETIC). There are other, non-ribosomal peptide biosynthesis (PEPTIDE BIOSYNTHESIS, NUCLEIC ACID-INDEPENDENT) mechanisms carried out by PEPTIDE SYNTHASES and PEPTIDYLTRANSFERASES. Further modifications of peptide chains yield functional peptide and protein molecules.
A method which uses specific precipitation reactions to separate or collect substances from a solution.
A galanin receptor subtype with high affinity for GALANIN-LIKE PEPTIDE and low affinity for full length GALANIN and galanin peptide fragments.
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