Tyrosine-based "activatable pro-tag": enzyme-catalyzed protein capture and release

Biotechnology and Bioengineering
Angela T LewandowskiWilliam E Bentley

Abstract

Protein recovery is often achieved by a series of capture and release steps that often involve chromatographic binding and elution. We report an alternative, non-chromatographic, capture and release approach that employs enzymes and the stimuli-responsive polysaccharide chitosan. We capture our protein using the enzyme tyrosinase that oxidizes accessible tyrosine residues of the protein and "activates" these residues for covalent capture (i.e., conjugation) onto chitosan. Using fusions of green fluorescent protein (GFP) we observed that: (i) enzymatic activation is required for protein capture to chitosan; and (ii) capture is enhanced (approximately five-fold) by engineering the protein to have a penta-tyrosine fusion tag that provides additional accessible tyrosine residues for enzymatic activation. Because the fusion tag appears to be the primary site for capture, and capture requires activation, we designate penta-tyrosine as a "pro-tag." The captured GFP-chitosan conjugate possesses the pH-responsive solubility that is characteristic of chitosan. We exploit this pH-responsive solubility to facilitate purification of the captured protein. Two enzymatic methods were explored to release the captured GFP from the chitosan conju...Continue Reading

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Citations

Jul 16, 2008·Biomedical Microdevices·Xiaolong LuoGary W Rubloff
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Nov 7, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Shinichi Sato, Hiroyuki Nakamura
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