Purification of Proteins using Peptide-ELP Based Affinity Precipitation.

07:00 EST 21st December 2019 | BioPortfolio

Summary of "Purification of Proteins using Peptide-ELP Based Affinity Precipitation."

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.


Journal Details

This article was published in the following journal.

Name: Journal of biotechnology
ISSN: 1873-4863


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