With an expanding life-science sector and growing production of recombinant proteins, the need for efficient downstream processing is increasing. Certain proteins are sensitive to the harsh conditions often used in protein purification, such as low pH, which can result in aggregation and denaturation. ZCa is a domain derived from Protein A that can be used for calcium-dependent purification of antibodies without the need for acidic pH. Based on this domain, the CaRA library has been constructed, which targets other therapeutic proteins than human antibodies. Four of the proteins isolated from the CaRA library, namely CaRA_scFv_1, CaRA_scFv_2, CaRA_G-CSF_1 and CaRA_G-CSF_3, are presented here for the purification of single chain variable fragment and granulate colony stimulating factor. The four proteins were produced as monomers, trimers and hexamers in an attempt to increase the binding capacity and attached to a matrix for purification using site-specific coupling. The successful binders CaRA_scFv_1 and CaRA_scFv_2 showed high affinity for their target protein scFv and were able to selectively capture an increased number of molecules through multimerization. Calcium-dependent binding was demonstrated by elution at neutral pH using the calcium chelator citrate, thus concluding that these multimerized CaRA variants can be used to considerably increase the efficiency in scFv purification while providing excellent purity and significantly reducing the risk of aggregation.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:kth-309515 |
| Date | January 2021 |
| Creators | Öst, Linnea |
| Publisher | KTH, Proteinvetenskap |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | TRITA-CBH-GRU ; 2021:275 |
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