Development of a Fast Organic Extraction-Precipitation Method for Improved Purification of Elastin-Like Polypeptides That Is Independent of Sequence and Molecular Weight.

ABSTRACT: Elastin-like polypeptides (ELP), an increasingly popular tag for protein purification, commonly rely upon inverse transition cycling (ITC) to exploit their lower critical solution temperature characteristics for purification. While considerably faster than chromatography, ITC is still time consuming and often fails to remove host cell contaminants to an acceptable level for in vivo experiments. Here, we present a rapid purification workflow for ELP of broadly varying molecular weight and sequence using a polar organic solvent extraction and precipitation strategy. Four different ELP purification methods were directly compared for their ability to remove host cell protein, nucleic acids, and lipopolysaccharide (LPS) contaminants using a model ELP. On the basis of these findings, an optimized extraction-precipitation method was developed that gave highly pure ELP from bacterial pellets in approximately 2.5 h while removing major host cell contaminants, including LPS to levels below 1 EU/mL, to produce highly pure material that is suitable for in vivo applications. Application of this method to the rapid purification of an ELP-epidermal growth factor fusion gave an isolate that retained its capacity to bind to epidermal growth factor receptor positive cells, thereby demonstrating that this method is capable of producing a functional construct after purification by organic extraction-precipitation.

SUBMITTER: Sweet C 

PROVIDER: S-EPMC8496954 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature