Elastin-like polypeptide-intein mediated protein purification

BIOT 437

Alison R. Gillies, agillies@princeton.edu, Department of Chemical Engineering, Princeton University, A-217 Engineering Quadrangle, Olden St., Princeton University, Princeton, NJ 08544 and David W. Wood, dwood@princeton.edu, Departments of Chemical Engineering and Molecular Biology, Princeton University, Engineering Quadrangle, A213 Olden Street, Princeton, NJ 08544.
An alternative to conventional affinity-tag separations has been developed for the purification of recombinant proteins. This method replaces the affinity resins found in conventional systems with elastin-like polypeptide tags. In this system, an ELP gene is attached to a gene for a self-cleaving protein linker, and the two are then joined to the product protein gene. The resulting expressed fusion protein is then purified via salt and temperature-dependent aggregation of the ELP tag, combined with sequential rounds of centrifugation and resuspension to wash the product protein. Self-cleavage of the ELP tag is induced by a pH shift and the purified target protein is recovered following precipitation of the cleaved ELP tag. This method has been very successful in E. coli. In principle, the ELP system will be applicable to a wide range of expression systems and hosts, and early modeling suggests applications in biologics manufacturing at very large scale.
 

Downstream Processing: Non-Chromatographic Separation Techniques: Improving Process Throughput
8:00 AM-11:00 AM, Thursday, August 23, 2007 BCEC -- 107B, Oral

Division of Biochemical Technology

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007