Establishing Novel Immunoaffinity Purification Methods for Fusion Proteins

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Establishing Novel Immunoaffinity Purification Methods for Fusion Proteins

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Title: Establishing Novel Immunoaffinity Purification Methods for Fusion Proteins
Author: Kirouac, Courtney Elyse ORCID  0000-0002-6647-5389
Citation: Kirouac, Courtney Elyse. 2016. Establishing Novel Immunoaffinity Purification Methods for Fusion Proteins. Master's thesis, Harvard Extension School.
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Abstract: The experiments in this thesis were performed to determine if novel uses of three fusion proteins could be established as a means of improving the protein purification process, and in particular, the elution step, thus resulting in the establishment of novel immunoaffinity purification methods.

There are numerous fusion tags currently available for use as purification tools. Many of these current methods for protein purification require harsh elution steps, such as a low pH elution, which can be harmful to the protein. There are few purification methods that successfully purify protein, while maintaining a gentle pH environment for the protein.

The goal was to employ a new in-house tag, and enhance two previously established tags, to optimize purification methods that would not require low pH elution steps. For each fusion tag in this work, there were three major aspects to the establishment of the purification method. The first aspect was the expression and purification by Nickel-IMAC of the fusion protein. The second aspect was the expression, purification, and antibody selection of an antibody specific for the fusion tag. The third aspect was the purification of the fusion protein on an immunoaffinity column to which the antibody was immobilized.

The first fusion tag was a peptide-tag derived from the C-terminus of Protein Y. It was to be used as a Flag™-tag replacement. An antibody was selected based on ELISA results and a Protein A-bound antibody small-scale purification test. The peptide-fusion protein was successfully purified in PBS, by competitive elution with excess synthetic peptide, using the selected antibody in an immunoaffinity column format. Though column capacity was low, yet equivalent to an anti-Flag resin, it did result in pure protein.

The second fusion tag was GFP. An already established fusion tag, it is sensitive to low pH, therefore mutations were made to an anti-GFP antibody in order to convey pH-dependent binding. The antibody was chosen based on Octet Kinetic Analysis and Protein A-bound antibody small-scale purification tests. The GFP-fusion protein was successfully purified in non-extreme pH conditions using the selected antibody in an immunoaffinity column format. The capacity was better than that of the previous method, though the purity may have been slightly less.

The last fusion tag was a ZZ-tag, which is also a previously established tag. A synthetic version of the B-domain of Protein A, it was used for its ability to bind murine IgG1 in particular conditions, and its weaker affinity for murine IgG1 in PBS. After yielding difficult to interpret results from Octet Kinetic Analysis, a number of murine IgG1s were tested as possible immunoaffinity antibodies in an immunoaffinity format. The low capacity and recovery made it difficult to support this method for future use.

The successful fusion protein purification by two methods in neutral pH conditions offers two new tools for purification of difficult to purify and pH-sensitive proteins, as well as a new tool for better purification of lysates. In particular, these methods will be useful for Biogen, as the established immunoaffinity antibodies are available in-house, providing low-cost options, as well as less harsh options, for future protein purification projects.
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