Strategies for folding of affinity tagged proteins using GroEL and osmolytes

Abstract

Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide 1) if further stabilization of the folded product is required and 2) if the protein construct in question will ever be competent to fold with osmolytes.

Escherichia coli Thioredoxin-like Protein YbbN Contains an Atypical Tetratricopeptide Repeat Motif and Is a Negative Regulator of GroEL

“…In this study YbbN bound GroEL more strongly than any other detected protein in E. coli lysate, as judged by its elution behavior from a YbbN affinity resin. Poorly folded proteins often bind to GroEL tenaciously during overexpression and purification (42), and we cannot rule out that YbbN samples a partially unstructured conformation that binds to GroEL in this experiment. However, His 6 -tagged YbbN did not co-purify with large amounts of GroEL during purification, contrary to the expected copurification of the two proteins if YbbN were partially unstructured and strongly binding to GroEL as a substrate, suggesting that YbbN does not act as a substrate for GroEL under these conditions.…”

Protein folding and stability in the presence of osmolytes

“…It should be noted that osmolytes may be capable of regulating the activity of the molecular chaperones GroEL, DnaK, and ClpB in a concentration-dependent manner [78][79][80].…”

Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine

“…Many essential proteins require the assistance of molecular chaperones to fold correctly without the risk of aggregation in a crowded cellular environment [25,26]. The insoluble human mitochondrial PEPCK was solubilized to biologically active form by using GroEL and GroES chaperonins [35]. Presence of ethanol in low concentration in the culture media decreases the formation of IBs as studied in human proinsulin protein [36].…”