Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags

Biotechnology and Bioengineering
Alison R GilliesDavid W Wood

Abstract

We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimizat...Continue Reading

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Citations

May 8, 2010·Applied Microbiology and Biotechnology·Skander Elleuche, Stefanie Pöggeler
Jan 13, 2012·Protein Engineering, Design & Selection : PEDS·Yingping WuWenyan Wu
Jul 1, 2009·Applied Microbiology and Biotechnology·Roelof SchipperusFrits A de Wolf
Apr 3, 2010·Trends in Biotechnology·Baley A FongDavid W Wood
Oct 15, 2010·Biotechnology Progress·Wan-Yi WuDavid W Wood
Dec 21, 2010·Protein Expression and Purification·Wan-Yi WuDavid W Wood
Aug 24, 2013·Protein Expression and Purification·Tiana D WarrenDavid W Wood
Nov 26, 2009·Current Protocols in Protein Science·Wan-Yi WuDavid W Wood

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