Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

Abstract

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

Natural Products for Drug Discovery in the 21st Century: Innovations for Novel Drug Discovery

“…Complex natural compounds with potential target proteins will have to be evaluated properly to identify all its potential target proteins. One of the most utilised methods to identify target proteins and their biological activities is affinity chromatography [ 117 , 118 , 119 , 120 , 121 , 122 , 123 ]. This method is a pull-down method in which the natural product is immobilized on a physical solid support [ 124 ].…”

In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli

“…To purify recombinant proteins that are fused to solubility tags, two consecutive steps of affinity chromatography are often necessary. The first chromatographic procedure purifies the fusion protein and, following site‐specific proteolysis, a second subtractive chromatographic procedure is generally implemented to remove the cleaved tag and the site‐specific protease (Wang et al, 2015). Donnelly and collaborators used the CIP approach to obtain untagged proteins without depending on a second chromatographic step (Donnelly et al, 2006).…”